Nitric oxide donors for treatment of disease and injury

ABSTRACT

A method of promoting neurogenesis by administering a therapeutic amount of a phosphodiesterase inhibitor compound is a patient in need of neurogenesis promotion. A compound for providing neurogenesis having an effective amount of a phosphodiesterase inhibitor sufficient to promote neurogenesis. A phosphodiesterase inhibitor for promoting neurogenesis. A method of augmenting the production of brain cells and facilitating cellular structual and receptor changes by administering an effective amount of a phosphodiesterase inhibitor compound to a site in need of augmentation. A method of increasing both neurological and cognitive function by administering an effective amount of a phosphodiesterase inhibitor compound to a patient.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to treatments of disease and injury. Morespecifically, the present invention relates to methods and compoundsincluding nitric oxide donors and cell therapy for the treatment ofdisease and injury.

2. Background Art

Stroke is the third most common cause of death in the adult populationof the United States, and is a major cause of disability. Stroke occurswhen a section of the brain becomes infarcted, resulting in death ofbrain tissue from interruption of cerebral blood supply. Cerebralinfarcts associated with acute stroke cause sudden and dramaticneurological impairment. Other neurological diseases also result in thedeath of tissue and neurological impairment.

Pharmacological interventions have attempted to maximize the blood flowto stroke affected brain areas that might be able to survive, butclinical effectiveness has proven elusive. As stated in Harrison'sPrinciples of Internal Medicine (9^(th) Ed., 1980, p. 1926), “despiteexperimental evidence that . . . [cerebral vasodilators] increase thecerebral blood flow, as measured by the nitrous oxide method, they havenot proved beneficial in careful studies in human stroke cases at thestage of transient ischemic attacks, thrombosis-in-evolution, or in theestablished stroke. This is true of nicotinic acid, Priscoline, alcohol,papaverine, and inhalation of 5% carbon dioxide. . . . In opposition tothe use of these methods is the suggestion that vasodilators are harmfulrather than beneficial, since by lowering the systemic blood pressurethey reduce the intracranial anastomotic flow, or by dilating bloodvessels in the normal parts of the brain they steal blood from theinfarct.”

Additionally, diseases of the cardiovascular system are a leadingworldwide cause of mortality and morbidity. For example, heart failurehas been increasing in prevalence. Heart failure is characterized by aninability of the heart to deliver sufficient blood to the various organsof the body. Current estimates indicate that over 5 million Americanscarry the diagnosis of heart failure with nearly 500,000 new casesdiagnosed each year and 250,000 deaths per year attributed to thisdisease. Despite significant therapeutic accomplishments in the past twodecades, heart failure continues to increase in incidence reachingepidemic proportions and representing a major economic burden indeveloped countries.

Heart failure is a clinical syndrome characterized by distinctivesymptoms and signs resulting from disturbances in cardiac output or fromincreased venous pressure. Moreover, heart failure is a progressivedisorder whereby the function of the heart continues to deteriorate overtime despite the absence of adverse events. Thus, due to heart failure,inadequate cardiac output results.

Generally, there are two types of heart failure. Right heart failure isthe inability of the right side of the heart to pump venous blood intopulmonary circulation. A back-up of fluid in the body occurs and resultsin swelling and edema. Left heart failure is the inability of the leftside of the heart to pump blood into systemic circulation. Back-upbehind the left-ventricle then causes accumulation of fluid in thelungs.

The main resulting effect of heart failure is fluid congestion. If theheart becomes less efficient as a pump, the body attempts to compensatefor it by using hormones and neural signals, for example, to increaseblood volume.

Heart failure has numerous causes. For example, disease of heart tissueresults in dead myocardial cells that no longer function. Progression inleft ventricular dysfunction has been attributed, in part, to ongoingloss of these cardiomyocytes.

There have been numerous methods of treating and preventing heartfailure. For example, stem cells have been used to regenerate cardiaccells in acute cardiac ischemia and/or infarction or injury in animalmodels. In one particular example, viable marrow stromal cells isolatedfrom donor leg bones were culture-expanded, labeled, and then injectedinto the myocardium of isogenic adult rat recipients. After harvestingthe hearts from 4 days to 12 weeks after implantation, the implantationsites were examined and it was found that implanted stromal cells showthe growth potential in a myocardial environment. (Wang, et. al.)

Cardiomyocytes have been shown to differentiate in vitro frompluripotent embryonic stem (ES) cells of line D3 via embryo-likeaggregates (embryoid bodies). The cells were characterized by thewhole-cell patch-clamp technique, morphology, and gene expressionanalogy during the entire differentiation period. (Maltsev, et. al.,1994) Additionally, pluripotent mouse ES cells were capable todifferentiate into cardiomyocytes expressing major features of mammalianheart (Maltsev, et. al., 1993).

Stem cells, regardless of their origin (embryonic, bone marrow, skeletalmuscle, etc.), have the potential to differentiate into various, if notall, cell types of the body. Stem cells are able to differentiate intofunctional cardiac myocytes. Thus, the development of stem cell-basedtherapies for treating heart failure has many advantages over existingconventional therapies.

Accordingly, there is a need for a method of treating patients havingdisease or injury by combining cell therapy and the use of a nitricoxide donor.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a method ofpromoting neurogenesis by administering a therapeutic amount of aphosphodiesterase inhibitor compound to a patient in need ofneurogenesis promotion. Also provided is a compound for providingneurogenesis having an effective amount of a phosphodiesterase inhibitorsufficient to promote neurogenesis. A phosphodiesterase inhibitor forpromoting neurogenesis is also provided. Further, a method of augmentingthe production of brain cells and facilitating cellular structural andreceptor changes by administering an effective amount of aphosphodiesterase inhibitor compound to a site in need of augmentationis provided. There is provided a method of increasing both neurologicaland cognitive function by administering an effective amount of aphosphodiesterase inhibitor compound to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

Other advantages of the present invention are readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanying drawingswherein:

FIGS. 1A-D show cerebral vascular perimeters;

FIGS. 2A-C show proliferated cerebral endothelial cells;

FIGS. 3A-C show DETANONOate induces angiogenesis, as analyzed withthree-dimensional images;

FIGS. 4A-E show DETANONOate induces in vitro angiogenesis;

FIG. 5 shows a bar graph that shows quantitative data ofSildenafil-induced capillary-like tube formation;FIGS. 6A-I arephotographs showing the effects of treating cells with sildenafil;

FIGS. 7A and B are graphs that show levels of cGMP in the cerebellum andcortex respectively after treatment with sildenafil versus controls innonischemic rats;

FIG. 8 is a graph that shows localized CBF in rats treated withsildenafil versus controls;

FIGS. 9A and B are graphs that show the results of the adhesive-removaltest and mNSS test respectively;

FIGS. 10A and B are a photograph and graph respectively that shows theresults of treatment of BrdU positive cells in the SVZ using the therapyof the present invention;

FIGS. 11A and B are a photograph and graph respectively that shows theresults of treatment of BrdU positive cells in the vessels using thetherapy of the present invention;

FIGS. 12A-C are photographs that show that the treatment of the presentinvention induces endothelial tube formation by brain-derivedendothelial cells compared with controls;

FIG. 13 is a graph that shows that the treatment of the presentinvention increased VEGF secretion compared to controls;

FIGS. 14A-G are photographs shows the results of the therapy of thepresent invention;

FIGS. 15A-C are bar graphs that show the number of BrdU immunoreactivecells in the dentate gyrus (FIG. 15A), in the SVZ (FIG. 15B), and in theOB (FIG. 15C) in non-ischemic young adult rats at 14 (

) and 42 (

) days after treatment with DETA/NONOate or saline;

FIGS. 16A-C are bar graphs that show the number of BrdU immunoreactivecells in the dentate gyrus (FIG. 16A), in the SVZ (FIG. 16B), and in theOB (FIG. 16C) in non-ischemic aged rats at 14 (

) and 42 (

) days after treatment with DETA/NONOate or saline;

FIGS. 17A-D show the effect of SNAP treatment on infarct volume (FIG.17A), rotarod (FIG. 17B) and adhesive removal (FIG. 17C) tests as wellas animal body weight (FIG. 17D); and

FIGS. 18A andB are photographs that show RT-PCR of PDE5A1 (FIG. 18A) andPDE5A2 (FIG. 18B) mRNA in the cortex of non-ischemic rats (N in FIG. 18Aand FIG. 18B) and the ipsilateral cortex of rats 2 hours to 7 days afterischemia.

DESCRIPTION OF THE INVENTION

Generally, the present invention provides a method and compound fortreating injury or disease in multi-organ systems with a combination ofcellular therapy and a nitric oxide donor or PDE inhibitor. Thiscombination therapy increases the effectiveness of both therapieswithout increasing any risk to a patient. The benefit of the therapy isthat is augments organ plasticity by inducing neurogenesis,angiogenesis, and alterations in parenchymal cell structure andfunction. Additionally, because of the synergistic effect, lower dosesof each therapy can be given, thereby limiting any side effects orharmful effects of the drugs which can otherwise manifest themselves.Alternatively, the PDE inhibitor alone can be administered fortreatment.

By “PDE inhibitor” it is meant a compound that inhibits PDE. An exampleof such a compound is sildenafil (Viagra™). A PDE inhibitor is an agentthat reduces (e.g. selectively reduces) or eliminates the activity of aphosphodiesterase, such as PDE1-10 (e.g. type V phosphodiesterase, type10 phosphosdiesterase), and any other phosphodiesterases. In the contextof the methods and compositions of the present invention, thephosphodiesterase inhibitors include salts, esters, amides, prodrugs andother derivatives of the active agents (e.g. the PDE). Thephosphodiesterase inhibitor amplifies the effects of any NO produced.The Phosphodiesterase inhibitor can be used to produce vasodilation andimprovement in vascular function.

Examples of these inhibitor compounds include, but are not limited torolipram,, theophylline, pentoxifylline, cGMP, zaprinast, IBMX,milrinone,5-(2-ethoxy-5-morpholinoacetylphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,5-(5-morpholinoacetyl-2-n-propoxyphenyl)-1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-d]pyrimidin-7-one,5-[2-ethoxy-5-(4-methyl-1-piperazinylsulfonyl)-phenyl]1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-d]pyrimidin-7-one,5-[2-allyloxy-5-(4-methyl-1-piperazinylsulfonyl)-phenyl)-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,5-[2-ethoxy-5-[4-(2-propyl)-1-piperazinylsulfonyl)-phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-d]pyrimidin-7-one,5-[2-ethoxy-5-[4-(2-hydroxyethyl)-1-piperazinylsulfonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-d]pyrimidin-7-one,5-[5-[4-(2-hydroxyethyl)-1-piperazinylsulfonyl]-2-n-propoxyphenyl]-1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-d]pyrimidin-7-one,5-[2-ethoxy-5-(4-methyl-1-piperazinylcarbonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-dlpyrimidin-7-one,and5-[2-ethoxy-5-(1-methyl-2-imidazolyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7-H-pyrazolo[4,3-d]pyrimidin-7-one.

The phosphodiesterase inhibitors also can include griseolic acidderivatives, 2-phenylpurinone derivatives, phenylpyridone derivatives,fused and condensed pyrimidines, pyrimidopyrimidine derivatives, purinecompounds, quinazoline compounds, phenylpyrimidinone derivative,imidazoquinoxalinone derivatives or aza analogues thereof,phenylpyridone derivatives, and others. Specific examples of thephosphodiesterase inhibitors include1,3-dimethyl-5-benzylpyrazolo[4,3-d]pyrimidine-7-one,2-(2-propoxyphenyl)-6-purinone,6-(2-propoxyphenyl)-1,2-dihydro-2-oxypyridine-3-carboxamide,2-(2-propoxyphenyl)-pyrido[2,3-d]pyrimid4(3H)-one,7-methylthio-4-oxo-2-(2-propoxyphenyl)-3,4-dihydro-pyrimido[4,5-d]pyrimidine,6-hydroxy-2-(2-propoxyphenyl)pyrimidine-4-carboxamide,1-ethyl-3-methylimidazo[1,5a]quinoxalin-4(5H)-one,4-phenylmethylamino-6-chloro-2-(1-imidazoloyl)quinazoline,5-ethyl-8-[3-(N-cyclohexyl-N-methylcarbamoyl)-propyloxy]-4,5-dihydro-4-oxo-pyrido[3,2-e]-pyrrolo[1,2-a]pyrazine,5′-methyl-3′-(phenylmethyl)-spiro[cyclopentane-1,7′(8′H)-(3′H)-imidazo[2,1b]purin]4′(5′H)-one,1-[6-chloro-4-(3,4-methylenedioxybenzyl)-aminoquinazolin-2-yl)piperidine-4-carboxylicacid,(6R,9S)-2-(4-trifluoromethyl-phenyl)methyl-5-methyl-3,4,5,6a,7,8,9,9a-octahydrocyclopent[4,5]-midazo[2,1-b]-purin-4-one,1t-butyl-3-phenylmethyl-6-(4-pyridyl)pyrazolo[3,4-d]-pyrimid-4-one,1-cyclopentyl-3-methyl-6-(4-pyridyl)-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimid-4-one,2-butyl-1-(2-chlorobenzyl)6-ethoxycarbonylbenzimidaole, and2-(4-carboxypiperidino)-4-(3,4-methylenedioxybenzyl)amino-6-nitroquinazoline,and 2-phenyl-8-ethoxycycloheptimidazole.

Still other type V phosphodiesterase inhibitors useful in conjunctionwith the present invention include: IC-351 (ICOS);4-bromo-5-(pyridylmethylamino)-6-[3-(4-chlorophenyl)propoxy]-3(2H)pyridazinone;1-[4-[(1,3-benzodioxol-5-ylmethyl)amiono]-6-chloro-2-quinazolinyl]-4-piperidine-carboxylicacid, monosodium salt;(+)-cis-5,6a,7,9,9,9a-hexahydro-2-[4-(trifluoromethyl)-phenylmethyl-5-methylcyclopent-4,5]imidazo[2,1-b]purin-4(3H)one;furazlocillin;cis-2-hexyl-5-methyl-3,4,5,6a,7,8,9,9a-octahydrocyclopent[4,5]imridazo[2,1-b]purin-4-one;3-acetyl-1-(2-chlorobenzyl)-2-propylindole-6-carboxylate;4-bromo-5-(3-pyridylmethylamino)-6-(3-(4-chlorophenyl)propoxy)-3-(2H)pyridazinone;1-methyl-5-(5-morpholinoacetyl-2-n-propoxyphenyl)-3-n-propyl-1,6-dihydro-7H-pyrazolo(4,3-d)pyrimidin-7-one;1-[4-[(1,3-benzodioxol-5-ylmethyl)amino]-6-chloro-2-quinazolinyl]-4-piperidinecarboxylicacid, monosodium salt; Pharmaprojects No. 4516 (Glaxo Wellcome);Pharmaprojects No. 5051 (Bayer); Pharmaprojects No. 5064 (Kyowa Hakko;see WO 96/26940); Pharmaprojects No. 5069 (Schering Plough); GF-196960(Glaxo Wellcome); and Sch-51866.

Other type V phosphodiesterase inhibitors include, but are not limitedto DMPPO (Eddahibi (1988) Br. J. Pharmacol., 125(4): 681-688), and1-arylnaphthalene lignan series, including1-(3-bromo-4,5-dimethoxyphenyl)-5-chloro-3-[4-(2-hydroxyethyl)-1-piperazinylcarbonyl]-2-(methoxycarbonyl)naphthalenehydrochloride (27q) (Ukita (1999) J. Med. Chem. 42(7): 1293-1305).

By “multi-organ systems” it is meant systems that affect multipleorgans. Such organs include, but are not limited to, heart, liver, andbrain.

By “nitric oxide donor” it is meant a compound that is able to donatenitric oxide or promote increase of nitric oxide. There are families ofcompounds that donate nitric oxide. Included among these compounds are:DETANONOate (DETANONO, NONOate or 1-substituteddiazen-1-ium-1,2-diolates are compounds containing the [N(O)NO]—functional group: DEA/NO; SPER/NO; DETA/NO; OXI/NO; SULFI/NO; PAPA/NO;MAHMA/NO and DPTA/NO), PAPANONOate, SNAP(S-nitroso-N-acetylpenicillamine), sodium nitroprusside, and sodiumnitroglycerine. There are compounds that promote the increase in nitricoxide, such as phosphodiesterase inhibitors and L-arginine.

By “promoting neurogenesis” as used herein, it is meant that neuralgrowth is promoted or enhanced. This can include, but is not limited to,new neuronal growth or enhanced growth of existing neurons, as well asgrowth and proliferation of parenchymal cells and cells that promotetissue plasticity. Neurogenesis also encompasses, but is not limited to,neurite and dendritic extension and synaptogenesis.

By “augmentation” as used herein, it is meant that growth is eitherenhanced or suppressed as required in the specific situation. Therefore,if additional neuron growth is required, the addition of a nitric oxidedonor increases this growth. Nitric oxide donors, or sources of nitricoxide, prime cerebral tissue to compensate for damage brought on byinjury, neurodegeneration, or aging by enhancing receptor activation andpromoting cellular morphological change and cellular proliferation.

By “neurological” or “cognitive” function as used herein, it is meantthat the neural growth in the brain enhances the patient's ability tothink, function, or more. Humans treated with nitric oxide haveincreased production of brain cells that facilitate improved cognition,memory and motor function. Further, patients suffering from neurologicaldisease or injury when treated with nitric oxide have improvedcognition, memory, and motor function.

The term “cell therapy” as used herein includes, but is not limited to,administering stem cells, a generalized mother cell whose descendantsspecialize into various cell types. The stem cells have various originsincluding, but not limited to, embryo, bone marrow, liver, stromal, fattissue, and other stem cell origins known to those of skill in the art.These stem cells can be administered or placed into the desired areas asthey naturally occur, or can be engineered in any manner known to thoseof skill in the art. Thus, through various genetic engineering methodsincluding, but not limited to, transfection, deletion, and the like, thestem cells can be engineered in order to increase their likelihood ofsurvival or for any other desired purpose.

The terms “enrich” or “enrichment” as used herein are meant to include,but are not limited to, to make rich or richer by the addition orincrease of some desirable quality or quantity of substance. In thepresent invention, enrichment occurs by the addition or increase of morefunctional cardiac cells within or around the myocardium.

The terms “repopulate” or “repopulating” as used herein are meant toinclude, but are not limited to, the addition or replenishment ofcardiac cells within or around the myocardium. These additionallyreinforce the activity of currently functioning cells. Thus, replacementand/or reinforcement of existing cardiac cells occurs.

The purpose of the present invention is to promote an improved outcomefrom neuronal injury, or other injury, by augmenting the effects of thetreatment, for example neurogenesis, and augmenting the cellular changesthat promote functional improvement. For example, patients sufferneurological and functional deficits after stroke, CNS injury andneurodegenerative disease. These findings provide a means to enhancebrain compensatory mechanism to improve function after CNS damage ordegeneration. The induction of neurons and cellular changes induced bynitric oxide administration will promote functional improvement afterstroke, injury, aging and degenerative disease. This approach can alsoprovide benefit to patients suffering from other neurological diseasesuch as, but not limited to, ALS, MS, and Huntington's disease.Additionally, the methods and compositions of the present invention canenhance the effectiveness of cell therapy.

Nitric oxide administered at propitious times after CNS injury promotesneurogenesis in brain and is able to facilitate neurogenesis. Nitricoxide can also augment the effectiveness of cell therapy. As an initialexperiment, DETA/NO was employed, a compound with a long half-life (˜50hours) which produces NO. Increased numbers of new neurons wereidentified when this compound was administered at and beyond 24 hoursafter onset of stroke. Preferably, the compounds of the presentinvention are administered directly to the site of injury. For example,the compounds can be administered orally, intraperitoneally,intravenously, or in any other manner known to those of skill in the artto provide the desired result. However, the compounds can beadministered systemically if necessary for treatment.

The experimental data included herein show that a pharmacologicalintervention designed to induce production of NO can promoteneurogenesis. Three compounds have been employed, DETANONOate, andsildenafil (Viagra™)) SNAP, these compounds have successfully inducedneurogenesis and improved functional outcome after stroke. The compoundused likely crosses the blood brain barrier. Neurogenesis is a majorlast goal in neuroscience research. Developing a way to promoteproduction of neurons opens up the opportunity to treat a wide varietyof neurological disease, CNS injury and neurodegeneration. It ispossible to augment the production of neurons in non-damaged brain, soas to increase function.

The market for a class of drugs that promotes the production of neuronsis vast. Nitric oxide donors, of which DETANONO is but one example,promote neurogenesis. Increasing neurogenesis translates into a methodto increase, improve neurological, behavioral and cognitive function,with age and after injury or disease.

In recent years it has become abundantly clear that one mechanism forthe deterioration of function in heart failure of any etiology is due,in part, to the ongoing death of heart muscle cells (Sabbah, 2000). Thesolution to this problem is to enrich or repopulate the myocardium withnew cardiac cells, which take the place of lost cells or provideadditional reinforcement of the currently function cardiac cells,thereby improving the pumping function of the failing heart. The presentinvention is based on the use of cells therapy to treat disease.Although stem cells have different origins (embryo, bone marrow, liver,fat tissue, etc.), their important common characteristic is that theyhave the potential to differentiate into various, if not all, cell typesof the body. As previously mentioned, stem cells have been shown to beable to differentiate into cardiac muscle cells. (Maltsev et al., 1993and 1994).

The present invention is advantageous over all currently existingtreatments. For example, currently, treatment of heart failure is basedprimarily on the use of drugs that interfere with neurohumoral systems.Additionally, surgical treatment exists that include hearttransplantation as well as the use of ventricular or bi-ventricularassisting devices. The advantages offered by the present invention isthe ability to treat heart failure by directly addressing the primarycause of the disease, namely, loss of contractile units. Re-populationof the myocardium with stem cells that differentiate into contractileunits that contribute to the overall function of the failing heart,therefore, is novel and goes to the center of the problem. Otheradvantages include absence of side effects that are often associatedwith the use of pharmacological therapy and absence of immune rejectionthat plagues heart transplantation or other organ transplants.

The present invention has the potential to replace many current surgicaltherapies and possibly even pharmacological therapies. Devices currentlyexist that allow delivery of stem cells to the failing heart usingcatheter-based approaches, thus eliminating the need for open chestsurgery. Additionally, the present invention is applicable in both thehuman medical environment and veterinary setting.

The present invention treats injury or disease and improves and/orrestores normal function. More specifically, the present invention isused to augment cell therapy thereby enabling cell therapies to functionmore effectively and efficiently. Function is increased by enrichingand/or repopulating the injured cells via transplanted stem cells thatdifferentiate into the injured cells, thereby increasing function. Thus,the increase of contractile units increases the function of the heart.Additionally, the stem cells can also be responsible for the release ofvarious substances such as trophic factors. Thus, for example, therelease of trophic factors induces angiogenesis (increase of the numberof blood vessels) in order to increase cardiac function and/or treatheart failure. Therefore, the stem cells operate to increase cardiacfunction and/or treat heart failure through various mechanisms otherthan just differentiating into functional cardiac muscle cells.

The general method of transplanting stem cells into the-myocardiumoccurs by the following procedure. The stem cells and the nitric oxidedonor or PDE inhibitor are administered to the patient. Theadministration can be subcutaneously, parenterally includingintravenous, intraarterial, intramuscular, intraperitoneally, andintranasal administration as well as with intrathecal and infusiontechniques.

By the term “stem cell” is meant any form of cell therapy, including,but not limited to, hematopoietic cells which are capable ofself-regeneration when provided to a human subject in vivo, and canbecome lineage restricted progenitors, which further differentiate andexpand into specific lineages. As used herein, “stem cells” refers tohematopoietic cells and not stem cells of other cell types. Further,unless indicated otherwise, “stem cells” refers to human hematopoieticstem cells.

The term “stem cell” or “pluripotent” stem cell are used interchangeablyto mean a stem cell having (1) the ability to give rise to progeny inall defined lineages, and (2) stem cells capable of fully reconstitutinga seriously immunocompromised host in all blood cell types and theirprogeny, including the pluripotent hematopoietic stem cell, byself-renewal.

Bone marrow is the soft tissue occupying the medullary cavities of longbones, some haversian canals, and spaces between trabeculae ofcancellous or spongy bone. Bone marrow is of two types: red, which isfound in all bones in early life and in restricted locations inadulthood (i.e. in the spongy bone) and is concerned with the productionof blood cells (i.e. hematopoiesis) and hemoglobin (thus, the redcolor); and yellow, which consists largely of fat cells (thus, theyellow color) and connective tissue.

As a whole, bone marrow is a complex tissue comprised of hematopoieticstem cells, red and white blood cells and their precursors, mesenchymalstem cells, stromal cells and their precursors, and a group of cellsincluding fibroblasts, reticulocytes, adipocytes, and endothelial cellswhich form a connective tissue network called “stroma”. Cells from thestroma morphologically regulate the differentiation of hematopoieticcells through direct interaction via cell surface proteins and thesecretion of growth factors and are involved in the foundation andsupport of the bone structure. Studies using animal models havesuggested that bone marrow contains “pre-stromal” cells that have thecapacity to differentiate into cartilage, bone, and other connectivetissue cells. (Beresford, J. N.: Osteogenic Stem Cells and the StromalSystem of Bone and Marrow, Clin. Orthop., 240:270, 1989). Recentevidence indicates that these cells, called pluripotent stromal stemcells or mesenchymal stem cells, have the ability to generate intoseveral different types of cell lines (i.e. osteocytes, chondrocytes,adipocytes, etc.) upon activation. However, the mesenchymal stem cellsare present in the tissue in very minute amounts with a wide variety ofother cells (i.e. erythrocytes, platelets, neutrophils, lymphocytes,monocytes, eosinophils, basophils, adipocytes, etc.), and, in an inverserelationship with age, they are capable of differentiating into anassortment of connective tissues depending upon the influence of anumber of bioactive factors.

As a result, the inventors have developed a process for isolating andpurifying human mesenchymal stem cells from tissue prior todifferentiation and then culture expanding the mesenchymal stem cells toproduce a valuable tool for musculoskeletal therapy. The objective ofsuch manipulation is to greatly increase the number of mesenchymal stemcells and to utilize these cells to redirect and/or reinforce the body'snormal reparative capacity. The mesenchymal stem cells are harvested ingreat numbers and applied to areas of tissue damage to enhance orstimulate in vivo growth for regeneration and/or repair, to improveimplant adhesion to various prosthetic devices through subsequentactivation and differentiation, enhance hemopoietic cell production,etc.

Along these lines, various procedures are contemplated by the inventorsfor transferring, immobilizing, and activating the culture expanded,purified mesenchymal stem cells at the site for repair, implantation,etc., including injecting the cells at the site of a skeletal defect,incubating the cells with a prosthesis and implanting the prosthesis,etc. Thus, by isolating, purifying and greatly expanding the number ofcells prior to differentiation and then actively controlling thedifferentiation process by virtue of their positioning at the site oftissue damage or by pretreating in vitro prior to their transplantation,the culture-expanded, undifferentiated mesenchymal stem cells can beutilized for various therapeutic purposes such as to elucidate cellular,molecular, and genetic disorders in a wide number of metabolic bonediseases, skeletal dysplasias, cartilage defects, ligament and tendoninjuries and other musculoskeletal and connective tissue disorders.

Along these same lines, various procedures are contemplated by theinventors for transferring, immobilizing, and activating the mesenchymalstem or progenitor cells at the site for repair, implantation, etc.,through the use of various porous ceramic vehicles or carriers,including injecting the cells into the location of injury.

The human mesenchymal stem cells can be obtained from a number ofdifferent sources, including plugs of femoral head cancellous bonepieces, obtained from patients with degenerative joint disease duringhip or knee replacement surgery, and from aspirated marrow obtained fromnormal donors and oncology patients who have marrow harvested for futurebone marrow transplantation. Although the harvested marrow was preparedfor cell culture separation by a number of different mechanicalisolation processes depending upon the source of the harvested marrow(i.e. the presence of bone chips, peripheral blood, etc.), the criticalstep involved in the isolation processes was the use of a speciallyprepared medium that contained agents which allowed for not onlymesenchymal stem cell growth without differentiation, but also for thedirect adherence of only the mesenchymal stem cells to the plastic orglass surface area of the culture dish. By producing a medium thatallows for the selective attachment of the desired mesenchymal stemcells that were present in the marrow samples in very minute amounts, itwas possible to separate the mesenchymal stem cells from the other cells(i.e. red and white blood cells, other differentiated mesenchymal cells,etc.) present in the bone marrow.

As indicated above, the complete medium can be utilized in a number ofdifferent isolation processes depending upon the specific type ofinitial harvesting processes used in order to prepare the harvested bonemarrow for cell culture separation. In this regard, when plugs ofcancellous bone marrow were utilized, the marrow was added to thecomplete medium and vortexed to form a dispersion which was thencentrifuged to separate the marrow cells from bone pieces, etc. Themarrow cells (consisting predominantly of red and white blood cells, anda very minute amount of mesenchymal stem cells, etc.) were thendissociated into single cells by passing the complete medium containingthe marrow cells through syringes fitted with a series of 16, 18, and 20gauge needles. It is believed that the advantage produced through theutilization of the mechanical separation process, as opposed to anyenzymatic separation process, was that the mechanical process producedlittle cellular change while an enzymatic process could produce cellulardamage particularly to the protein binding sites needed for cultureadherence and selective separation, and/or to the protein sites neededfor the production of monoclonal antibodies specific for saidmesenchymal stem cells. The single cell suspension (which was made up ofapproximately 50-100.times.10.sup.6 nucleated cells) was thensubsequently plated in 100 mm dishes for the purpose of selectivelyseparating and/or isolating the mesenchymal stem cells from theremaining cells found in the suspension.

When aspirated marrow was utilized as the source of the humanmesenchymal stem cells, the marrow stem cells (which contained little orno bone chips but a great deal of blood) were added to the completemedium and fractionated with Percoll (Sigma, St. Louis, Mo.) gradientsmore particularly described below in Example 1. The Percoll gradientsseparated a large percentage of the red blood cells and the mononucleatehematopoietic cells from the low density platelet fraction whichcontained the marrow-derived mesenchymal stem cells. In this regard, theplatelet fraction, which contained approximately 30-50.times.10.sup.6cells was made up of an undetermined amount of platelet cells,30-50.times.10.sup.6 nucleated cells, and only about 50-500 mesenchymalstem cells depending upon the age of the marrow donor. The low-densityplatelet fraction was then plated in the Petri dish for selectiveseparation based upon cell adherence.

In this regard, the marrow cells obtained from either the cancellousbone or iliac aspirate (i.e. the primary cultures) were grown incomplete medium and allowed to adhere to the surface of the Petri dishesfor one to seven days according to the conditions set forth in Example 1below. Since no increase in cell attachment was observed after the thirdday, three days was chosen as the standard length of time at which thenon-adherent cells were removed from the cultures by replacing theoriginal complete medium with fresh complete medium. Subsequent mediumchanges were performed every four days until the culture dishes becameconfluent which normally required 14-21 days. This represented10.sup.3-10.sup.4 fold increase in undifferentiated human mesenchymalstem cells.

The cells were then detached from the culture dishes utilizing areleasing agent such as trypsin with EDTA (ethylene diaminetetra-aceticacid) (0.25% trysin, 1 mM EDTA (1.times.), Gibco, Grand Island, N.Y.) ora chelating agent such as EGTA (ethylene glycol-bis-(2-amino ethylether) N,N′-tetraacetic acid, Sigma Chemical Co., St. Louis, Mo.). Theadvantage produced through the use of a chelating agent over trypsin wasthat trypsin could possibly cleave off a number of the binding proteinsof the mesenchymal stem cells. Since these binding proteins containrecognition sites, when monoclonal antibodies were to-be produced, achelating agent such as EGTA as opposed to trypsin, was utilized as thereleasing agent. The releasing agent was then inactivated and thedetached cultured undifferentiated mesenchymal stem cells were washedwith complete medium for subsequent use.

These results indicated that under certain conditions, culture expandedmesenchymal stem cells have the ability to differentiate into bone whenincubated as a graft in porous calcium phosphate ceramics. Although theinternal factors which influence the mesenchymal stem cells todifferentiate into bone as opposed to cartilage cells are not wellknown, it appears that the direct accessibility of the mesenchymal stemcells to growth and nutrient factors supplied by the vasculature inporous calcium phosphate ceramics, as opposed to the diffusion chamber,influenced the differentiation of the mesenchymal stem cells into bone.

As a result, the isolated and culture expanded mesenchymal stem cellscan be utilized under certain specific conditions and/or under theinfluence of certain factors, to differentiate and produce the desiredcell phenotype needed for tissue repair.

Administration of a single dose of mesenchymal stem cells can beeffective to reduce or eliminate the T cell response to tissueallogeneic to the T cells or to “non-self” tissue, particularly in thecase where the T lymphocytes retain their nonresponsive character (i.e.,tolerance or anergy) to allogeneic cells after being separated from themesenchymal stem cells.

The dosage of the mesenchymal stem cells varies within wide limits andis fitted to the individual requirements in each particular case. Ingeneral, in the case of parenteral administration, it is customary toadminister from about 0.01 to about 5 million cells per kilogram ofrecipient body weight. The number of cells used will depend on theweight and condition of the recipient, the number of or frequency ofadministrations, and other variables known to those of skill in the art.The mesenchymal stem cells can be administered by a route that issuitable for the tissue, organ or cells to be transplanted. They can beadministered systemically, i.e., parenterally, by intravenous injectionor can be targeted to a particular tissue or organ, such as bone marrow.The human mesenchymal stem cells can be administered via a subcutaneousimplantation of cells or by injection of stem cell into connectivetissue, for example muscle.

The cells can be suspended in an appropriate diluent, at a concentrationof from about 0.01 to about 5×10⁶ cells/ml. Suitable excipients forinjection solutions are those that are biologically and physiologicallycompatible with the cells and with the recipient, such as bufferedsaline solution or other suitable excipients. The composition foradministration must be formulated, produced and stored according tostandard methods complying with proper sterility and stability.

Although the invention is not limited thereof, mesenchymal stem cellscan be isolated, preferably from bone marrow, purified, and expanded inculture, i.e. in vitro, to obtain sufficient numbers of cells for use inthe methods described herein. Mesenchymal stem cells, the formativepluripotent blast cells found in the bone, are normally present at verylow frequencies in bone marrow (1:100,000) and other mesenchymaltissues. See, Caplan and Haynesworth, U.S. Pat. No. 5,486,359. Genetransduction of mesenchymal stem cells is disclosed in Gerson et al U.S.Pat. No. 5,591,625.

Unless otherwise stated, genetic manipulations are performed asdescribed in Sambrook and Maniatis, MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

A detailed description of the method and composition of the presentinvention is set forth in Appendix A included herewith and incorporatedby reference in its entirety. While specific embodiments are disclosedherein, they are not exhaustive and can include other suitable designsthat vary in design and methodologies known to those of skill in theart. Basically, any differing design, methods, structures, and materialsknown to those skilled in the art can be utilized without departing fromthe spirit of the present invention.

Methods:

General methods in molecular biology: Standard molecular biologytechniques known in the art and not specifically described weregenerally followed as in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (1989),and in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1989) and in Perbal, A Practical Guideto Molecular Cloning, John Wiley & Sons, New York (1988), and in Watsonet al., Recombinant DNA, Scientific American Books, New York and inBirren et al (eds) Genome Analysis: A Laboratory Manual Series, Vols.1-4 Cold Spring Harbor Laboratory Press, New York (1998) and methodologyas set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531;5,192,659 and 5,272,057 and incorporated herein by reference. Polymerasechain reaction (PCR) was carried out generally as in PCR Protocols: AGuide To Methods And Applications, Academic Press, San Diego, Calif.(1990). In-situ (In-cell) PCR in combination with Flow Cytometry can beused for detection of cells containing specific DNA and mRNA sequences(Testoni et al, 1996, Blood 87:3822.)

General methods in immunology: Standard methods in immunology known inthe art and not specifically described are generally followed as inStites et al.(eds), Basic and Clinical Immunology (8th Edition),Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi (eds),Selected Methods in Cellular Immunology, W.H. Freeman and Co., New York(1980).

Delivery of Therapeutics

The compound of the present invention is administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the compound of the presentinvention can be administered in various ways. It should be noted thatit can be administered as the compound or as pharmaceutically acceptablesalt and can be administered alone or as an active ingredient incombination with pharmaceutically acceptable carriers, diluents,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. The patient being treated is a warm-blooded animal and, inparticular, mammals including man. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

It is noted that humans are treated generally longer than the mice orother experimental animals exemplified herein which treatment has alength proportional to the length of the disease process and drugeffectiveness. The doses can be single doses or multiple doses over aperiod of several days, but single doses are preferred.

The doses can be single doses or multiple doses over a period of severaldays. The treatment generally has a length proportional to the length ofthe disease process and drug effectiveness and the patient species beingtreated.

When administering the compound of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, can also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S. Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

A pharmacological formulation of the compound utilized in the presentinvention can be administered orally to the patient. Conventionalmethods such as administering the compounds in tablets, suspensions,solutions, emulsions, capsules, powders, syrups and the like are usable.Known techniques which deliver it orally or intravenously and retain thebiological activity are preferred.

In one embodiment, the compound of the present invention can beadministered initially by intravenous injection to bring blood levels toa suitable level. The patient's levels are then maintained by an oraldosage form, although other forms of administration, dependent upon thepatient's condition and as indicated above, can be used. The quantity tobe administered will vary for the patient being treated and will varyfrom about 100 ng/kg of body weight to 100 mg/kg of body weight per dayand preferably will be from 10 mg/kg to 10 mg/kg per day.

EXAMPLE 1

The effects of NO on angiogenesis and the synthesis of vascularendothelial growth factor (VEGF) were investigated in a model of focalembolic cerebral ischemia in the rat. Compared to control rats, systemicadministration of an NO donor, DETANONOate, to rats 24 hours afterstroke significantly enlarged vascular perimeters, and increased thenumber of proliferated cerebral endothelial cells and the numbers ofnewly generated vessels in the ischemic boundary regions, as evaluatedby three-dimensional laser scanning confocal microscopy. Treatment withDETANONOate significantly increased VEGF levels in the ischemic boundaryregions as measured by ELISA. A capillary-like tube formation assay wasused to investigate whether DETANONOate increases angiogenesis inischemic brain via activation of soluble guanylate cyclase. DETANONOateinduced capillary-like tube formation was completely inhibited by asoluble guanylate cyclase inhibitor,1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ). Blocking VEGFactivity by a neutralized antibody against VEGF receptor 2 significantlyattenuated DETANONOate-induced capillary-like tube formation. Moreover,systemic administration of a phosphodiesterase type 5 inhibitor(Sildenafil) to rats 24 hours after stroke significantly increasedangiogenesis in the ischemic boundary regions. Sildenafil and an analogof cyclic guanosine monophosphate (cGMP) also induced capillary-liketube formation. These findings suggest that exogenous NO enhancesangiogenesis in ischemic brain, which is mediated by the NO/cGMPpathway. Furthermore, the data suggest that NO, in part via VEGF, canenhance angiogenesis in ischemic brain.

Treatment of stroke with nitric oxide (NO) donors reduces functionalneurological deficits. NO is a pleiotropic molecule that affects manyphysiological and pathophysiological functions. Animals treated with NOdonors evoke cell proliferation in neurogenic regions of the brain, suchas the subventricular zone and the dentate gyrus. However, themechanisms underlying the improvement of neurological function aftertreatment require clarification.

A potential therapeutic target for NO treatment of stroke isangiogenesis. Administration of proangiogenic agents, such as basicfibroblast growth factor (bFGF) and vascular endothelial growth factor(VEGF), to animals with stroke significantly reduce neurologicaldysfunction. Inrcubation of human vascular smooth muscle cells with NOdonors increases VEGF synthesis and the NO synthase (NOS) antagonistN^(W)nitro-I-arginine methyl ester (L-NAME) reduces VEGF generation.Endothelial NO synthase (eNOS) deficient mice exhibit significantimpairment of angiogenesis in the ischemic limb, indicating that NOmodulates angiogenesis in ischemic tissue. Thus, there appears to be acoupling between NO, VEGF and angiogenesis. However, there have been nostudies on the effects of NO donors on VEGF and angiogenesis afterstroke. Accordingly, the fact that NO increases VEGF and enhancesangiogenesis via a cyclic guanosine monophosphate pathway (cGMP) wastested in a model of focal embolic cerebral ischemia in the rat.

Materials and Methods:

Animal Model:

Male Wistar rats weighing 320-380 gm were employed. The middle cerebralartery (MCA) was occluded by placement of an embolus at the origin ofthe MCA.

Experimental Protocol: 1) To examine whether exogenous NO affectsneovascularization in ischemic animals,(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1,2-diolate(DETANONOate), an NO donor-with a half-life of 57 hours underphysiological conditions, was administered to ischemic rats. DETANONOate(0.4 mg/kg) was intravenously administered to rats (n=8) 24 hours afterstroke and daily (i.p) for an additional 6 consecutive days. Ischemicrats (n=8) treated with the same volume of decayed DETANONOate were usedas a control group. All rats were sacrificed 14 days after stroke. 2) Toexamine the effect of exogenous NO on brain levels of VEGF, DETANONOate(0.4 mg/kg) or saline was administered to ischemic rats (n=3 for eachgroup) with the identical paradigm described in Protocol 1. These ratswere sacrificed 7 days after stroke. 3) To examine whether increases incGMP promote angiogenesis in ischemic brain, a phosphodiesterase type 5(PDE 5) inhibitor which increases cGMP, Sildenafil dissolved in 3 ml oftap water (2 mg/kg), was fed to ischemic rats (n=8) at 24 hours afterstroke and daily for an additional 6 days. Rats were sacrificed 14 daysafter stroke.

Bromodeoxyuridine Labeling:

Bromodeoxyuridine (BrdU, Sigma Chemical), the thymidine analog that isincorporated into the DNA of dividing cells during S-phase, was used formitotic labeling. BrdU (50 mg/kg) was injected (i.p) daily for 13consecutive days into ischemic rats starting 1 day after MCA occlusion.

Three Dimensional Image Acquisition and Analysis:

To examine neovascularization in ischemic brain, fluoresceinisothiocyanate (FITC) dextran (2×10⁶ molecular weight, Sigma, St. Louis,Mo.; 0.1 ml of 50 mg/ml) was administered intravenously to the ischemicrats subjected to 14 days of MCAo. The brains were rapidly removed fromthe severed heads and placed in 4% of paraformaldehyde at 4° C. for 48hours. Coronal sections (100 μm) were cut on a vibratome. The vibratomesections were analyzed with a Bio-Rad MRC 1024 (argon and krypton)laser-scanning confocal imaging system mounted onto a Zeiss microscope(Bio-Rad; Cambridge, Mass.), as previously described. Seven 100 μm thickvibratome coronal sections at 2 mm intervals from bregma 5.2 mm tobregma −8.8 mm from each animal injected with FITC-dextran wereselected. Eight brain regions in the ipsilateral and contralateralhemispheres were selected within a reference coronal section (interaural8.8 mm, bregma 0.8 mm). These regions were scanned in 512×512 pixel(276×276 μm²) format in the x-y direction using a 4× frame-scan averageand twenty-five optical sections along the z-axis with a 1 μm step-sizewere acquired under a 40× objective. Vascular branch points, segmentlengths and diameters were measured in three dimensions using softwaredeveloped in the laboratory. Image acquisition and analysis wereperformed blindly.

Immunohistochemistry and Quantification:

For BrdU immunostaining, DNA was first denatured by incubating brainsections (6 μm) in 50% formamide 2×SSC at 65° C. for 2 hours and then in2N HCl at 37° C. for 30 minutes. Sections were then rinsed with trisbuffer and treated with 1% of H₂O₂ to block endogenous peroxidase.Sections were incubated with a mouse monoclonal antibody (mAb) againstBrdU (1:1000, Boehringer Mannheim, Indianapolis, Ind.) overnight andincubated with biotinylated secondary antibody (1:200, Vector,Burlingame, Calif.) for 1 hour.

To quantify BrdU immunoreactive endothelial cells, numbers ofendothelial cells and numbers of BrdU immunoreactive endothelial cellsin ten enlarged vessels adjacent to the ischemic lesion were countedfrom each rat. Numbers of endothelial cells and BrdU immunoreactiveendothelial cells in the ten vessels of the contralateral homologousarea were also counted. Data are presented as percentage of BrdUimmunoreactive endothelial cells to total endothelial cells in tenenlarged vessels from each rat.

Vascular perimeters were measured on coronal sections immunostained withan anti-von Willebrand factor antibody as previously described.

ELISA for VEGF:

The ischemic boundary regions and homologous tissue in the contralateralhemisphere were dissected. The tissue was homogenized and centrifuged at10,000 g for 20 min at 4° C. and the supernatant was collected. ELISAfor VEGF in the supernatants was performed using a commerciallyavailable kit specific for rat VEGF (R&D, systems) according to themanufacture's instruction.

Capillary-like Tube Formation Assay:

An in vitro angiogenesis assay was performed. Briefly, 0.8 ml of growthfactor reduced Matrigel (Becton Dickinson) was added to pre-chilled 35mm culture dishes and allowed to polymerize at 37° C. for 2 to 5 hours.Mouse brain-derived endothelial cells (2×10⁴ cells) were incubated for 3hours in Dulbecco's modified Eagle's medium (DMEM) containingDETANONOate, Sildenafil, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one(ODQ), 8-Br-cGMP, or a rat anti-mouse neutralizing antibody to VEGFreceptor 2 (VEGFR2, DC101, Imclone System). For quantitativemeasurements of capillary tube formation, three random areas of Matrigeldishes were imaged and the length of continuous cords of three or morecells was measured.

Statistical Analysis:

One way analysis of variance (ANOVA) followed by Student-Newman-Keulstest was used. The data were presented as means±SE. A value of p<0.05was taken as significant.

Results:

Effects of DETANONOate and Sildenafil on Angiogenesis In Vivo:

To examine whether exogenous NO enhances angiogenesis in ischemic brain,DETANONOate was administered to rats 24 hours after stroke for 7 days.Treatment with DETANONOate significantly (p<0.01) enlarged vascularperimeters (FIGS. 1A and 1D) around the ischemic lesion but did notenlarge vessels in the contralateral hemisphere (FIGS. 1B and 1D)compared with the ipsilateral vessels in the control rats (FIGS. 1C and1D). Endothelial cells in enlarged thin walled vessels exhibited BrdUimmunoreactivity (FIGS. 2A and 2B) and quantitative analysis revealedthat the numbers of proliferated endothelial cells significantly(p<0.05) increased in rats treated with DETANONOate (FIG. 2C). Tofurther examine angiogenesis, three dimensional analysis was performedusing software developed in the laboratory, which measures numbers ofsegments, segment lengths and diameters of vessels. Treatment withDETANONOate significantly (p<0.05) increased the numbers of capillarysegments in the boundary regions of ischemia (FIG. 3A and Table 1)compared with the numbers in ischemic rats treated with same volume ofdecayed DETANONOate (FIG. 3B and Table 1). The capillary segments in theDETANONOate treated groups exhibited significantly smaller diameters(FIG. 3A and Table 1) and shorter segment lengths (FIG. 3A and Table 1),suggesting that these are newly generated vessels. A significantincrease of angiogenesis was also detected in rats treated sildenafil(Table 1).

Effects of DETANONOate and Sildenafil on Brain Levels of VEGF:

To examine whether administration of DETANONOate increases brain levelsof VEGF, ELISA for endogenous rat VEGF was. performed. ELISAmeasurements revealed that treatment with DETANONOate significantly(p<0.05) increased VEGF levels in the ischemic boundary regions from13.4±1.5 μg/ml in the control group (n=3) to 28.9±1.0 μg/ml in theDETANONOate treated group (n=3). Since NO increases cGMP, induction ofVEGF by DETANONOate could occur via the cGMP pathway. PDE 5 is highlyspecific for hydrolysis of cGMP. Brain levels of VEGF in rats treatedwith the PDE 5 inhibitor, Sildenafil were measured. Treatment withSildenafil significantly (p<0.05) increased VEGF levels (34.4±2.9 μg/mlvs 13.4±1.5 μg/ml in the control, n=3 per group) in the ischemicboundary.

Effects of Soluble Guanylate Cyclase Inhibitor and Neutralization ofVEGFR2 on DETANONOate-induced Capillary-like Tube Formation:

To support the hypothesis that DETANONOate increases angiogenesis inischemic brain via the activation of soluble guanylate cyclase, theeffects of DETANONOate on angiogenesis were further analyzed using acapillary-like tube formation assay. A significant increase incapillary-like tube formation was detected when mouse brain-derivedendothelial cells were incubated with DETANONOate (0.2 μM, FIGS. 4B and4E) compared with the endothelial cells incubated with DMEM only (FIGS.4A and 4E). However, DETANONOate-induced capillary-like tube formationwas completely inhibited when the endothelial cells were incubated withDETANONOate in the presence of ODQ, a potent inhibitor of solubleguanylate cyclase (FIGS. 4C and 4E), indicating that the NO/cGMPsignaling pathway is involved in mediating the effects of DETANONOate onangiogenesis. To examine whether DETANONOate also enhances angiogenesisvia increases in VEGF, the endothelial cells were incubated for 3 hoursin the presence of DETANONOate (0.2 μM) and a rat anti-mouseneutralizing antibody to VEGFR2 (DC101, 10 μg/ml). The biologicalactivity of this antibody against VEGFR2 in the mouse has beendemonstrated. Treatment of endothelial cells with the antibody againstVEGFR2 significantly (P<0.05) reduced DETANONOate-induced capillary-liketube formation (FIGS. 4D and 4E), suggesting that VEGF is involved inDETANONOate-induced angiogenesis.

Effects of Sildenafil on Capillary-like Tube Formation:

Incubation of the endothelial cells with Sildenafil (100 to 500 nM)produced concentration-dependent capillary-like tube formation (FIG. 5).8-BrcGMP (1 mM), a stable analog of cGMP, also significantly (p<0.05)increased capillary-like tube formation (FIG. 5). ODQ (10 μM)significantly inhibited Sidenafil-induced capillary-like tube formation(FIG. 5), indicating that angiogenesis by Sildenafil is dependent onbasal activity of sGC in the endothelial cells. ODQ did notsignificantly inhibit 8-BrcGMP-induced capillary-like tube formation(FIG. 5), confirming that this effect is independent of solubleguanylate cyclase activation.

Discussion:

The major findings of the present study are that 1) administration ofDETANONOate or Sildenafil 24 hours after stroke increases synthesis ofVEGF and enhances angiogenesis in ischemic brain; 2) ODQ, an inhibitorof soluble guanylate cyclase, completely inhibits DETANONOate-inducedcapillary-like tube formation; 3) Sildenafil, an inhibitor of PDE5,induces capillary-like tube formation; and 4) blocking of VEGF activityby a neutralized antibody against VEGFR2 attenuates DETANONOate-inducedcapillary-like tube formation; Together, these data indicate thatexogenous NO enhances angiogenesis in ischemic brain via the NO/cGMPdependent pathway and an inhibitor of PDE 5 (Sildenafil) augmentsangiogenesis. The data also suggest a coupling of NO, VEGF andangiogenesis.

NO plays an important role in angiogenesis. However, there have been nostudies on the effect of NO on angiogenesis in ischemic brain. Micelacking eNOS exhibit severe impairment of spontaneous angiogenesis inresponse to limb ischemia, and administration of L-arginine acceleratesangiogenesis. In the present study, administration of DETANONOatesignificantly increased the numbers of enlarged vessels and proliferatedendothelial cells in the ischemic boundary regions, which is consistentwith data that NO induces vessel dilation and endothelial cellproliferation.

NO activates soluble guanylate cyclase, thereby producing an increase ofcGMP in target cells. PDE 5 enzyme is highly specific for hydrolysis ofcGMP and Sildenafil citrate is a potent inhibitor of PDE5 which causesintracellular accumulation of cGMP 22. DETA/NONOate-inducedcapillary-like tube formation was completely inhibited by ODQ, aselective inhibitor of soluble guanylate cyclase, suggesting thatDETA/NONOate enhances brain angiogenesis via activation of solubleguanylate cyclase. The results are in agreement with previous reportsthat NO activates soluble guanylate cyclase in angiogenesis. To obtainfurther evidence that increases in cGMP contribute to NO-enhancedangiogenesis in ischemic brain, the PDE 5 inhibitor (Sildenafil) wasadministered to rats 24 hours after stroke. The data show that treatmentwith Sildenafil enhances angiogenesis in the boundary regions ofischemia. Moreover, Sildenafil and 8-BrcGMP (an analog of cGMP) inducecapillary-like tube formation in a culture of brain derived endothelialcells. ODQ significantly inhibits Sildenafil- but not 8-BrcGMP-inducedcapillary-like tube formation, indicating this response is dependent onbasal activity of sGC. Therefore, the data support the conclusion thatthe NO/cGMP pathway mediates DETANONOate-induced angiogenesis inischemic brain.

VEGF mediates angiogenesis and NO and VEGF can interact to promoteangiogenesis. A high concentration of NO donor downregulates VEGFexpression in endothelial cells. In contrast, recent studies showendogenous NO enhances VEGF synthesis. The eNOS-deficient mice exhibitsignificant impairment of angiogenesis in the ischemic hindlimb andadministration of VEGF to these mice does not increase impairedangiogenesis, indicating that NO is a downstream mediator forVEGF-induced angiogenesis. Angiogenesis in response to VEGF depends onthe tissue microenvironments. The data show that exogenous NO increasedischemic brain levels of VEGF and blocking VEGF activity attenuatedDETANONOate-induced capillary-like tube formation, suggesting that NOinduces VEGF synthesis in brain and VEGF at least in part mediatesDETANONOate-induced angiogenesis. These findings are consistent withprevious studies that NO derived from NO donors can increase thesynthesis of VEGF. In addition, the PDE 5 inhibitor, Sildenafil,increases brain levels of VEGF in the ischemic brain, suggesting thatcGMP likely contributes to NO-induced VEGF synthesis. This finding isinconsistent with a previous study that the cGMP is not involved inNO-induced upregulation of VEGF in cultured human articularchondrocytes. The reason for this discrepancy can be attributed to celltype difference, but remains enigmatic.

Angiogenesis is tightly regulated by two families of growth factors, theVEGF and angiopoietin families, as well as endothelial cell interactionwith extracellular matrix. Upregulation of VEGF and angiopoietin genesare correlated with brain angiogenesis after stroke. Furthermore, strokeinduces expression of VEGF receptors 1 and 2 in endothelial cells ofcerebral vessels ¹². Administration of NO-donor could amplify endogenousVEGF in the astrocytes and endothelial cells and consequently increasedVEGF enhances angiogenesis in ischemic brain via interaction withupregulated VEGF receptors in the endothelial cells, as previouslydemonstrated that treatment with VEGF increases angiogenesis inexperimental stroke. Newly generated vessels function in ischemic brain,and they can contribute to functional recovery via improvement oflong-term perfusion. Therefore, the positive interaction between NO andVEGF suggests that combination treatment with an NO donor and VEGF canhave synergistic effects on angiogenesis.

FIG. 1 shows cerebral vascular perimeters. Treatment with DETANONOateenlarged cerebral vessels in the ischemic boundary (FIG. 1A), but notvessels in the homologous area of the contralateral hemisphere (FIG. 1B)from a representative rat. FIG. 1C shows an enlarged vessel in theischemic boundary from a representative rat treated with decayedDETANONOate. Quantitative data (FIG. 1D) shows that treatment of strokewith DETANONOate significantly increased vascular perimeters comparedwith the ipsilateral vascular perimeters in the control rats. *p<0.01 vsipsilateral. Bar in C=50 μm.

FIG. 2 shows proliferated cerebral endothelial cells. FIG. 2A showsseveral BrdU immunoreactive endothelial cells (arrows) in an enlargedthin-wall vessel of a representative rat treated with DETANONOate. FIG.2B shows a BrdU immunoreactive endothelial cell (arrow) in an enlargedvessel of a representative rat from the control group. Although ischemiainduced proliferation of endothelial cells (FIG. 2C, control), treatmentwith DETANONOate significantly increased the numbers of proliferatedendothelial cells (FIG. 2C, DETANONO). *p<0.01 vs the contralateralhemisphere and #p<0.05 vs the ipsilateral hemisphere in the controlgroup. Bar in B=10 μm.

FIG. 3 shows DETANONOate induces angiogenesis, as analyzed withthree-dimensional images. Computer-generated images were originallyderived from images obtained with three-dimensional laser scanningconfocal microscopy. Treatment with DETANONOate increased the numbers ofnewly generated vessels (FIG. 3A), compared with the numbers of newvessels in rats in the control group (FIG. 3B). However, DETANONOate didnot alter vascular morphology in the contralateral hemisphere (FIG. 3C).Green and red colors in the images represent vascular diameters largerand smaller than 7.5 μm, respectively. Image size is 276×276×25 μm³ andunit in the images is μm.

FIG. 4 shows DETANONOate induces in vitro angiogenesis. Mouse brainderived endothelial cells were incubated with DMEM for 3 hours in theabsence of DETANONOate (FIG. 4A), in the presence of DETANONOate (0.2μM, FIG. 4B), and in the presence of DETANONOate with ODQ (FIG. 4C) orwith an antibody against VEGFR2 (FIG. 4D). Capillary-like tube formationwas induced by DETANONOate (FIG. 4B), and this effect was inhibited byODQ (FIG. 4C) or by the antibody against VEGFR2 (FIG. 4D). Similarresults were obtained in at least four experiments. A bar graph (FIG.4E) shows quantitative data of capillary-like tube formation. *p<0.05 vscontrol and #p<0.05 vs DETANONOate (0.2 μM). NO 0.1 and 0.2 representDETANONOate 0.1 and 0.2 μM. DC101 represents the antibody againstVEGFR2.

FIG. 5 shows a bar graph shows quantitative data of Sildenafil-inducedcapillary-like tube formation. Sildenafil (100-500 nM) and 8-BrcGMPinduced capillary-like tube formation and ODQ significantly inhibitedSildenafil (300 nM) induced capillary-like tube formation but did notattenuate 8-BrcGMP-induced capillary-like tube formation. *p<0.05 vscontrol and #p<0.05 vs Sildenafil 300 nM. Sil.=Sildenafil.

EXAMPLE 2

Methods

Male Wistar rats were subjected to embolic middle cerebral arteryocclusion. Sildenafil (Viagra) was administered orally for 7 consecutivedays starting 2 or 24 hours after stroke onset at doses of 2 or 5 mg/kgper day. Ischemic rats administered the same volume of tap water wereused as a control group. Functional outcome tests (foot-fault, adhesiveremoval) were performed. Rats were killed 28 days after stroke foranalysis of infarct volume and newly generated cells within thesubventricular zone and the dentate gyrus. Brain cGMP levels, expressionof PDE5, and localized cerebral blood flow were measured in additionalrats.

Results

Treatment with sildenafil significantly (P=0.05) enhanced neurologicalrecovery in all tests performed. There was no significant difference ofinfarct volume among the experimental groups. Treatment with sildenafilsignificantly (P=0.05) increased numbers ofbromodeoxyuridine-immunoreactive cells in the subventricular zone andthe dentate gyrus and numbers of immature neurons, as indicated byIII-tubulin (TuJ1) immunoreactivity in the ipsilateral subventricularzone and striatum. The cortical levels of cGMP significantly increasedafter administration of sildenafil, and PDE5 mRNA was present in bothnonischemic and ischemic brain.

Conclusions

Sildenafil increases brain levels of cGMP, evokes neurogenesis, andreduces neurological deficits when given to rats 2 or 24 hours afterstroke. These data show that this drug that is presently in the clinicfor sexual dysfunction has a role in promoting recovery from stroke.

Nitric oxide (NO) is a potent activator of soluble guanylate cyclase andcauses cGMP formation in target cells. Phosphodiesterase type 5 (PDE5)enzyme is highly specific for hydrolysis of cGMP and is involved inregulation of cGMP signaling. Sildenafil is a novel inhibitor of PDE5and causes intracellular accumulation of cGMP. Administration of an NOdonor to rats with stroke significantly increases brain levels of cGMP,induces cell genesis, and improves functional recovery. Functionalrecovery is partly due to increases in levels of cGMP resulting fromadministration of an NO donor. Therefore, administration of sildenafil,a PDE5 inhibitor, to rats subjected to stroke enhances improvement ofneurological outcome during stroke recovery.

Materials and Methods

Sildenafil is a weak basic compound, which is therefore only partiallyionized at physiological pH and has a half-life of 0.4 hour in rats. Afilm tablet of Viagra (content 100 mg sildenafil, purchasedcommercially) was weighed and powdered.

Animal Model

Male Wistar rats weighing 320 to 380 g were used in the present study.The middle cerebral artery (MCA) was occluded by placement of an embolusat the origin of the MCA.

Experimental Protocols

The experiments were performed to examine whether administration ofsildenafil affects cell proliferation and neurological behavior,sildenafil at 2 mg/kg (n=10) or 5 mg/kg (n=9) dissolved in 3 mL of tapwater was administered orally to rats 2 hours after MCA occlusion anddaily for an additional 6 days. Another group of the ischemic rats(n=10) was treated orally with sildenafil (2 mg/kg) 24 hours after MCAocclusion and daily for an additional 6. days. Ischemic rats (n=9) weretreated with the same volume of tap water as a control group. Functionaltests were performed and body weight was measured before ischemia and at4, 7, 14, 21, and 28 days after onset of MCA occlusion. All rats werekilled 28 days after MCA occlusion. Experiments were also performed toexamine whether administration of sildenafil affects brain-cGMP levels,nonischemic rats were treated with sildenafil at 2 mg/kg (n=6) or tapwater (n=10) for 7 days. These rats were killed 1 hour after the lasttreatment for measurements of brain levels of cGMP. Experiments werealso performed to examine the effects of sildenafil on cerebral bloodflow (CBF) and blood pressure, nonischemic rats (n=6) were treatedorally with sildenafil, and local CBF and mean arterial blood pressurewere measured starting at 30 minutes and continuing for 180 minutesafter administration of sildenafil. Experiments were also performed toexamine brain PDE5, nonischemic rats and ischemic rats were killed at 2,4, 24, 48, and 168 hours after the onset of ischemia (n=3 for each timepoint). Reverse transcription (RT)-polymerase chain reaction (PCR) wasperformed to detect PDE5 in brain tissue.

cGMP Measurement in Brain Tissue

Levels of cGMP were measured with the use of a commercially availablelow-pH immunoassay kit (R&D Systems Inc). The sensitivity of the assaywas approximately 0.6 μmol/mL for the nonacetylated procedure. The brainwas rapidly removed, and the cortex and the cerebellum were separated.Brain tissue was weighed and homogenized in 10 volumes of 0.1N HClcontaining 1 mmol/L 3-isobutyl-1-methylxanthine.

RT-PCR Analysis

To examine the presence of PDE5 in rat brain tissue, primers for PDE5A1and PDE5A2 were synthesized according to published sequence. The 5′primer 5′-AAAACTCGAGCAGAAACCCGCGGCA-AACACC-3′ and the 3′ primer5′-GCATGAGGACTTTGAG-GCAGAGAGC-3′ amplified a cDNA fragment coding forN-terminal regions of rat PDE5A1. The 5′ primer5′-ACCTCTGCTATGTTGCCCTTTGC-3′ and the 3′ primer5′-GCATGAGGACTTTGAGGCAGAGAGC-3′ amplified a cDNA fragment coding to ratPDE5A2. For cDNA synthesis, total RNA extracted from brain tissue wasreverse transcribed. Samples were denatured at 95° C. for 2 minutes andthen amplified for 40 cycles. Each cycle consisted of denaturation at95° C. for 30 seconds, annealing at 62° C. for 1 minute, and extensionat 72° C. for 2 minutes. The samples (30 μL per well) wereelectrophoresed on 1.5% agarose containing ethidium bromide.

Body Weight Loss

Animals were weighed before and at 4, 7, 14, 21, and 28 days afterembolic ischemia. Body weight loss is presented as a percentage ofpreischemic body weight.

Foot-Fault Test

Rats were tested for placement dysfunction of forelimbs with themodified foot-fault test before ischemia and at 4, 7, 14, 21, and 28days after embolic ischemia. Rats were set on an elevated hexagonal gridof different sizes and placed their paws on the wire while moving alongthe grid. With each weight-bearing step, the paw may fall or slipbetween the wire. The total number of steps (movement of each forelimb)that the rat used to cross the grid was counted, and the total number offoot-faults for each forelimb was recorded.

Adhesive Removal Test

An adhesive removal test was used to measure somatosensory deficits andwas performed before MCA occlusion and at 4, 7, 14, 21 and 28 days afterMCA occlusion.

Bromodeoxyuridine Labeling

Bromodeoxyuridine (BrdU) was used to measure cell proliferation. Animalsreceived daily intraperitoneal injections of BrdU (50 mg/kg; Sigma) onthe day of stroke and subsequently for 14 consecutive days. Cellproliferation in the subventricular zone and dentate gyrus was measuredin rats killed at 28 days (in experimental protocol 1, all 4 groups)after ischemia.

Immunohistochemistry

For BrdU immunostaining, DNA was first denatured by incubating brainsections (6 m) in 50% formamide 2′ SSC at 65° C. for 2 hours and then in2N HCl at 37° C. for 30 minutes. Sections were then rinsed with Trisbuffer and treated with 1% of H2 O2 to block endogenous peroxidase.Sections were incubated with a primary antibody to BrdU (1:100) at roomtemperature for 1 hour and then incubated with biotinylated secondaryantibody (1:200, Vector) for 1 hour. Reaction product was detected withthe use of 3′3′-diaminobenzidine-tetrahydrochloride (DAB; Sigma). For'III-tubulin (TuJ1) immunostaining, which identifies immature neurons,12 coronal sections were incubated with the antibody against TuJ1(1:1000) at 4° C. overnight and were then incubated with a biotinylatedhorse anti-mouse immunoglobulin antibody at room temperature for 30minutes. Double immunofluorescent staining for BrdU and TuJ1 wasperformed to determine whether BrdU-immunoreactive cells expressneuronal phenotype on the coronal sections.

Image Analysis and Quantification

Measurements of BrdU-immunoreactive cells were performed onparaffin-embedded 6-4m-thick sections.11 BrdU-immunostained sectionswere digitized with the use of a 40 objective (Olympus BX40) via theMCID computer imaging analysis system (Imaging Research).BrdU-immunoreactive nuclei were counted on a computer monitor to improvevisualization and in 1 focal plane to avoid oversampling. AllBrdU-immunoreactive-positive nuclei were counted in both the ipsilateraland contralateral walls of the lateral ventricle of the subventricularzone and in the dentate gyrus. For the subventricular zone, every 40thcoronal section was selected from each rat for a total of 7 sectionsbetween anteroposterior 10.6 mm of the genu corpus callosum andanteroposterior 8.74 mm of the anterior commissure crossing. For thedentate gyrus, every. 50th coronal section was selected from each ratfor a total of 8 sections between anteroposterior 5.86 mm andanteroposterior 2.96 mm of the granule cell layer. BrdU-immunoreactivenuclei in the subventricular zone and in the dentate gyrus are presentedas the number of the cells per square millimeter (mean SE). Densityvalues for the 7 sections (subventricular zone) and 8 sections (dentategyrus) were averaged to obtain a mean density value for each animal.Numbers of TuJ1-immunoreactive cells were counted in the subventricularzone and striatum, and data are presented as the number ofTuJ1-immunoreactive cells per section (mean SE).

Monitoring of Relative Erythrocyte Flow Velocity

Relative erythrocyte flow velocity was measured by laser-Dopplerflowmetry (PeriFlux PF4 flowmeter; Perimed AB) in the tissue under thelaser-Doppler flowmetry probe.13 A burr hole 1.5 mm in diameter wasdrawn on the skull 2 mm posterior to the bregma and 6 mm lateral tomidline.13 The dura was left intact. After the application of mineraloil onto the burr hole, the probe was placed 0.5 mm above the duralsurface. Relative flow velocities were measured 30 minutes afteradministration of sildenafil. This measurement reflects relativelylocalized CBF.14 Values of flow velocities are presented as a percentageof the contralateral hemispheric values.

Measurements of Infarct Volume

Measurement of infarct volume was measured on 7 hematoxylin andeosin-stained coronal sections with the use of a Global Laboratory Imageanalysis program (Data Translation). Briefly, the area of bothhemispheres and the infarct area (mm 2) were calculated by tracing thearea on the computer screen. Infarct volume (mm 3) was determined bymultiplying the appropriate area by the section interval thickness. Theinfarct volume is presented as the percent-age of infarct volume of thecontralateral hemisphere (indirect volume calculation).

Statistical Analysis

For analysis of neurological functional recovery and body weight, thegeneralized estimation equations (GEE) analysis approach was usedinstead of ANOVA because the data did not meet assumptions of normalityand equal variance for ANOVA. A paired t test or signed rank test wasused to test the difference in cell proliferation between ipsilateraland contralateral regions of subventricular zone, dentate gyrus, andstriatum. The GEE analysis approach was used to study the treatmenteffect on cell proliferation in the ipsilateral and contralateralsubventricular zone regions, dentate gyrus, and striatum. All values arepresented as mean SE. Statistical significance was set at P 0.05.

Results

Effects of Sildenafil on Cell Proliferation

Ischemic rats treated with sildenafil (2 or 5 mg/kg) initiated at 2 or24 hours after stroke had significant (P 0.05) increases in numbers ofBrdU-immunoreactive cells in the dentate gyrus of both hemispheres(Table 1) compared with control rats. Treatment with sildenafil at adose of 2 mg/kg (at 2 or 24 hours) significantly (P 0.05) increasednumbers of BrdU-immunoreactive cells in the ipsilateral subventricularzone (Table 1), and the 5 mg/kg dose (at 2 hours) significantly (P 0.05)increased numbers of BrdU-immunoreactive cells in the subventricularzone of both hemispheres (Table 1) compared with numbers ofBrdU-immunoreactive cells in control rats.

FIG. 6 shows the effect of treatment with sildenafil increasedTuJ1-immunoreactive cells 28 days after ischemia. FIG. 6A is a samplefrom a representative rat, robust increases in numbers ofTuJ1-immunoreactive cells in the ipsilateral subventricular zonecompared with the contralateral subventricular zone (FIG. 6B) are shown.Ependymal cells (arrows in FIGS. 6A and B) were not TuJ1 immunoreactive.TuJ1-immunoreactive cells exhibited cluster in the ipsilateral striatum(FIG. 6C) compared with the homologous tissue in the contralateralhemisphere (FIG. 6D). Double immunostaining with anti-bodies againstTuJ1 and BrdU shows that BrdU-immunoreactive cells (FIGS. 6E and G,green, arrows) were TuJ1 immunoreactive (FIGS. 6E and F, red, arrows).FIG. 6E is a merged image from FIGS. 6F and G. FIGS. 6H and I showquantitative data of numbers of TuJ1-immunoreactive cells in thesubventricular zone (n 6 in each group) and striatum (n 6 in eachgroup), respectively. (*P=0.05, **P=0.01, #P=0.05 vs control group. LVindicates lateral ventricle. Bars 10 m in FIGS. 1B and G and 20 m in C.)TABLE 1 Density of Newborn Cells in Brain Subventricular Zone DentateGyrus Groups Ipsilateral Contralateral Ipsilateral ContralateralSildenafil 2 mg/kg, 2 h 383 ± 23.44* 296 ± 19.74 55 ± 3.99* 55 ± 2.10†Sildenafil 5 mg/kg, 2 h 437 ± 32.97† 312 ± 23.79* 59 ± 5.26* 58 ± 5.38*Sildenafil 2 mg/kg, 24 h 374 ± 16.07* 295 ± 24.54 57 ± 4.21* 56 ± 4.76*Control 295 ± 32.69 246 ± 18.54 44 ± 2.96 42 ± 3.01Density of newborn cells is presented as mean ± SEM number ofBrdU-immunoreactive cells per mm².*P < 0.05,†P < 0.01 vs control group.Effects of Sildenafil on Immature Neurons

Administration of sildenafil robustly increased number ofTuJ1-immunoreactive cells in the ipsilateral subventricular zone (FIG.6A) and striatum (FIG. 6C). TuJ1-immunoreactive cells exhibited clustersin the ipsilateral striatum (FIG. 6C). Some of the TuJ1-immunoreactivecells were BrdU immunoreactive (FIGS. 6E to 6G). Quantitativemeasurements revealed that administration of sildenafil at a dose of 2or 5 mg/kg significantly (P=0.05) increased numbers ofTuJ1-immunoreactive cells in the ipsilateral and contralateralsubventricular zones compared with the number in control rats (FIG. 6H).Treatment with sildenafil also significantly increased the number ofTuJ1 cells in the ipsilateral striatum compared with homologous tissuein the contralateral hemisphere and in the ipsilateral striatum ofcontrol rats (FIG. 6I).

Effects of Sildenafil on Neurological Outcome

The ischemic rats treated with sildenafil at a dose of 2 or 5 mg/kgsignificantly improved performance on the foot-fault test (Table 2) andthe adhesive removal test (Table 3) during 4 to 21 days compared withcontrol rats when treatment was initiated at 2 hours after onset ofischemia. In addition, treatment with sildenafil at doses of 2 and 5mg/kg significantly reduced animal body weight loss (Table 4). Incontrast, infarct volumes measured 28 days after ischemia were notsignificantly different among these groups (Table 5), suggesting thatinfarct volume does not contribute to improvement of functionalrecovery. Sildenafil was also administered at a dose of 2 mg/kg to theischemic rats starting at 24 hours after onset of ischemia. Ischemicrats receiving sildenafil exhibited significant (P=0.05) improvements atthe foot-fault (Table 2) and adhesive removal (Table 3) tests during 7to 28 days after stroke. Rats treated with sildenafil also showed asignificant (=0.05) reduction in body weight loss at 4, 7, 14, 21, and28 days after ischemia (Table 4). However, there were no significantdifferences in infarct volume between ischemic animals treated withsildenafil and animals in the control group (Table 5).

Effects of Sildenafil on cGMP

The cerebellar levels of cGMP (FIG. 7A, control) were higher than thecortical (FIG. 7B, control) levels in nonischemic control rats, which isconsistent with previous studies. 4 Treatment with sildenafil at a doseof 2 or 5 mg/kg for 7 days significantly (P=0.05) increased the corticallevels of cGMP (FIG. 7B) compared with levels in the control group.

Effects of Sildenafil on Localized CBF

Administration of sildenafil at a dose of 2 mg/kg to nonischemic ratssignificantly increased localized CBF levels compared with the controlrats (FIG. 8). Significantly increased localized CBF persisted for 70minutes after administration of sildenafil (FIG. 8).

PDE5 in Rat Brain

RT-PCR analysis revealed both PDE5A1 (257 bp) and PDE5A2 (149 bp)transcripts in non-ischemic rat brain tissue, indicating the presence ofPDE5 (data not shown). Levels of PDE5A1 and PDE5A2 mRNA measured by banddensity (n=3 for each time point) did not show a statistical differenceafter MCA occlusion compared with the nonischemic rats.

Discussion

The present study demonstrates that treatment of focal cerebral ischemiain rats with sildenafil significantly improved recovery of neurologicaloutcome and significantly increased numbers of BrdU- andTuJ1-immunoreactive cells in ischemic brain. In addition, administrationof sildenafil significantly increased cortical levels of cGMP.Therefore, the data show that increased cGMP levels resulting fromadministration of sildenafil mediates enhanced neurological outcome.TABLE 2 Foot-Fault Test % of Foot-Faults Groups Before Ischemia 4 d 7 d14 d 21 d 28 d Sildenafil 2 mg/kg, 2 h  1.1 ± 0.01 22.81 ± 3.1 15.2 ±1.6† 13.2 ± 1.2†  9.1 ± 1.5†  8.2 ± 1.4 Sildenafil 5 mg/kg, 2 h 1.02 ±0.02  17.9 ± 2.9 16.6 ± 1.4* 14.6 ± 2.1*  9.5 ± 1.6†  7.6 ± 1.4Sildenafil 2 mg/kg, 24 h 1.03 ± 0.03  25.3 ± 3.8 14.4 ± 1.0† 10.0 ± 0.5† 9.0 ± 0.5†  5.3 ± 0.8* Control 1.06 ± 0.07  31.4 ± 3.4 24.9 ± 3.0 22.0± 2.6 19.4 ± 2.7 11.8 ± 1.9Values are mean ± SE for specified number of days after ischemia.*P < 0.05,†P < 0.01 vs control group.

TABLE 3 Adhesive Removal Test Seconds Groups Before Ischemia 4 d 7 d 14d 21 d 28 d Sildenafil 2 mg/kg, 2 h  7.0 ± 0.1  96.4 ± 9.8 41.9 ± 8.4†27.6 ± 3.9† 23.6 ± 5.1* 15.9 ± 3.8 Sildenafil 5 mg/kg, 2 h 16.7 ± 0.4100.7 ± 9.2 70.7 ± 10.8* 38.6 ± 7.9† 26.0 ± 6.6† 14.8 ± 3.9 Sildenafil 2mg/kg, 24 h  6.8 ± 0.3   102 ± 6.6 49.4 ± 4.5† 14.1 ± 1.3† 14.0 ± 1.0†10.7 ± 0.9 Control  7.0 ± 0.3 114.7 ± 3.6 95.7 ± 5.2 67.8 ± 9.6 43.4 ±5.7 19.0 ± 3.3Values are mean ± SE.*P < 0.05,†P < 0.01 vs control group.

PDE5 is an important enzyme for the hydrolysis of cGMP. The observationsof PDE5 mRNA in the cortex in nonischemic rats are consistent withprevious studies in which PDE5 mRNA and proteins were detected in rats.Sildenafil citrate is a potent inhibitor of PDE5 and causesintracellular accumulation of cGMP. The data show that administration ofsildenafil significantly increased brain cGMP levels. in parallel withthe findings, local administration of zaprinast, a relatively selectiveinhibitor of PDE5, to rat brain slices leads to an increase of cGMPrelease. Thus, the data indicate that sildenafil affects brain PDE5.cGMP modulates vasorelaxing effects in vascular muscle. Administrationof sildenafil transiently increased CBF in nonischemic rats, consistentwith previous in vitro and in vivo studies. Administration of zaprinastelicits dilatation of the basilar artery in rats and produces dilatationof dog cerebral arteries. Administration of sildenafil at a dose of5-mg/kg decreases the systolic arterial blood pressure, and the effectlasts for at least 6 hours. However, the effects of sildenafil on CBF donot provide neuroprotection because the treatment did not reduce infarctvolume and the treatment was effective even when sildenafil was firstadministered at 24 hours after the onset of ischemia, which is farbeyond the therapeutic window for neuroprotection.

Another new finding of the present study is that treatment withsildenafil significantly increases proliferation of progenitor cells inthe subventricular zone and the dentate gyrus and numbers of immatureneurons, as assayed by TuJ1 immunostaining. Administration ofDETA/NONOate, an NO donor, significantly enhances neurogenesis. NOactivates soluble guanylate cyclase and leads to formation of cGMP,while sildenafil inhibits PDE5 activity and results in inhibition ofcGMP breakdown. Taken together, these data show that cGMP regulatesneurogenesis. The findings are consistent with previous studies thatcGMP-dependent protein kinase type I enhances sensory neuron precursorproliferation. It is interesting to note that neuronal progenitor cellsin the subventricular zone migrate to the olfactory bulb, and afterreaching the olfactory bulb, they differentiate into mature neurons.These data are consistent with the observation that formation ofolfactory memory is mediated by cGMP concentration. cGMP levels inneurons are also involved in the modulation of dendritic and axonalguidance. Increased intracellular cGMP via sema can convert dendriticand axonal guidance from repulsion to attraction. In addition, cGMPenhances neurite outgrowth in hippocampal neurons in culture and in PC12cells. Furthermore, aged rats exhibit a decrease in the basal levels ofcGMP as a consequence of a more active degradation of cGMP by aphosphodiesterase in the aged brain compared with the adult brain.Decreases in NO and cGMP synthesis in aged brain can have importantfunctional implications in the processes of learning and memory.Neurogenesis can translate into functional improvement. For example,mice with a high rate of neurogenesis in the dentate gyrus exhibitenhanced performance on a hippocampal-dependent task, whereas ade-creasing rate of neurogenesis is correlated with impairment on such atask. Therefore, enhancement of neurogenesis can contribute tofunctional recovery after treatment with sildenafil. In summary, theresults of this study demonstrate that administration of sildenafilafter stroke enhances functional recovery and augments neurogenesis inthe rat.

FIG. 7 shows that levels of cGMP in the cerebellum (FIG. 7A) and cortex(FIG. 7B) after treatment with sildenafil in nonischemic rats (n=6);n=10 in control group. TABLE 4 Animal Body Weight Loss % of PreischemicBody Weight Groups Before Ischemia 4 d 7 d 14 d 21 d 28 d Sildenafil 2mg/kg, 2 h 100 81.8 ± 2.3 86.6 ± 2.5† 89.6 ± 4.7* 98.2 ± 3.7† 105.2 ±3.0* Sildenafil 5 mg/kg, 2 h 100 84.6 ± 1.8 81.8 ± 2.5* 84.7 ± 3.9 95.2± 3.5* 101.3 ± 4.3 Sildenafil 2 mg/kg, 24 h 100 86.9 ± 0.2 84.9 ± 1.8†89.2 ± 2.6* 99.5 ± 2.0* 107.2 ± 2.5* Control 100 74.7 ± 1.1 73.8 ± 1.177.6 ± 3.4 81.5 ± 4.7  92.4 ± 4.6Values are mean ± SE.*P < 0.05,†P < 0.01 vs control group.

TABLE 5 Infarct Volume Groups Infarct Volume, % Sildenafil 2 mg/kg, 2 h35.2 ± 3.3 Sildenafil 5 mg/kg, 2 h 37.7 ± 4.3 Sildenafil 2 mg/kg, 24 h35.5 ± 0.9 Control 38.3 ± 1.7Infarct volume is presented as mean ± SE percentage of lesion relativeto the contralateral hemisphere.

EXAMPLE 3

Male Wistar rats (n=32) were subjected to middle cerebral arteryocclusion (MCAo) and were randomized with 8 rats in four treatmentgroups, with treatment initiated 1 day after stroke. Groups included: 1)phosphate buffered saline (PBS); 2) subtherapeutic DETA-NONOate (NN) ata dose of 0.4 mg/kg (IP); 3) subtherapeutic hMSCs (1×10⁶ cells-iv); and4) combination subtherapeutic NN and hMSCs. Functional outcomemeasurements consisted of a Neurologic Severity Scale (18 point scale)(NSS) and the Adhesive Removal Test performed prior to stroke,immediately before treatment and at 7 and 14 days after treatment. Datawere well balanced among groups before the treatment (p-values>0.30).hMSC by NONO interaction was observed at 1.4 days (p-value=0.86).However, there was an overall hMSC effect on NSS at 14 days. Rats withthe treatment of hMSC+NONO had a significant improvement on NSS at 14days compared to rats in the control group (p-value=0.01), while rats inthe low dose hMSC had a borderline improvement on NSS at 14 days(pvalue=0.05) compared to rats in the control. No significant differenceon NSS at 14 days was detected between the control and NONO treatedgroups (p=0.64) and between the hMSC treated and the hMSC+NONO treatedgroups (p=0.48). The same treatment effects were observed onAdhesive-Removal test score at 14 days; rats treated with the hMSC+NONOhad a significant improvement at 14 days compared to rats in the controlgroup (p-value=0.01). There was a borderline improvement at 14 days inrats treated with the low dose (i.e. subtherapeutic) of hMSC alone andno significant improvement in rats treated with subtherapeutic NONOalone compared to the rats in controls with p-values of 0.06 and 0.64respectively. At 7 days, the neurological functional improvement wasobserved only on NSS for the rats treated with the combination of hMSCand NONO compared to rats in the control group (p-value=0.03). Thesedata indicate that combination of subtherapeutic therapeutic modalitiesof hMSCs and an NO donor (DETA-NONOate) significantly improvesfunctional outcome compared to control PBS treatment animals.

Volume of Cerebral Infarction and the Presence of MSCs in IschemicBrain:

No significant reduction of volume of ischemic damage was detected inrats with hMSC (30.7±6.2%) or NONOate (32.2±6.2%) and combination hMSCswith NONOate (28.7±6.7%) treatment, compared with control rats subjectedto MCAo with PBS (34.9±7.4%). hMSCs were identifiedimmunohistochemically using an antibody specific for human chromosomes(MAB1281). Within the brain tissue, cells derived from hMSCs werecharacterized by MAB1281 staining. No MAB1281 positive cells were foundin the non-hMSCs treated rats. MSCs identified by MAB1281 survived andwere distributed throughout the damaged brain of recipient rats. MAB1281positive cells were observed in multiple areas, including cortex andstriatum of the ipsilateral hemisphere. The vast majority of MAB1281positive hMSCs were located in the ischemic boundary zone. Few cellswere observed in contralateral hemisphere. There was no significantincrease in numbers of MAB1281 cells between the hMSC and combinationtherapy groups. These data indicate that the volume of cerebralinfarction is not affected by the combination therapy and that thenumbers of MSCs that enter brain is not altered by the coadministrationof an NO donor.

Neurogenesis:

BrdU (50 mg/kg-ip) was injected daily for 14 days after treatment in allgroups. BrdU is a thymidine analog that labels newly formed DNA andthereby identifies newly formed cells. FIG. 9 shows that in theipsilateral hemisphere subventricular zone, BrdU positive cells weresignificantly increased in the hMSC (2b, 40.6±10.7) or/and NONOate (FIG.9 c, 43.6±10.0/section; FIG. 9 d, 67.4±22.8/section) treated groupcompared to the control PBS treatment group (FIG. 9 a, 29.8±8.8/section)(p<0.05). BrdU found in the cytoplasm of macrophage-like cells were notcounted. Double staining shows that the BrdU positive cells express theneuronal markers NeuN, neuron specific enolase (NSE) and the astrocytemarker GFAP. The percentage of BrdU reactive cells that express NeuN andGFAP proteins was approximately, 3%, 3% and 6%, respectively. These dataindicate that that while individual subtherapeutic NO donor and MSCtherapy failed to significantly increase neurogenesis compared to PSCcontrol treated animals, combination therapy significantly promotesneurogenesis in ischemic brain.

Angiogenesis:

Enlarged and thin walled vessels are termed “mother” vessels and havebeen found under conditions of cerebral ischemic angiogenesis. FIG. 10shows that enlarged vessels exhibited a significant (p<0.05) increase inBrdU immunoreactive endothelial cells (FIG. 10 a) in hMSCs treatmentgroup and NONOate treatment groups compared with control MCAO group inthe ipsilateral hemisphere. BrdU reactive endothelial cells weresignificantly increased in the ipsilateral hemisphere of the combinationsubtherapeutic hMSCs/NONOate treatment group compared with theipsilateral hemisphere of hMSCs or NONOate alone treatment groups (FIG.10 b, p<0.05). These data indicate that combination NO donor and MSCtherapy significantly increases angiogenesis compared with theindividual therapies.

Enhanced angiogenesis after combination therapy is also demonstrated inFIG. 11 which shows three-dimensional images of cerebral vessels in theischemic penumbra after MCAO following the treatment 1) PBS; 2) NONOate;3) hMSCs; 4) hMSCs+NONOate. FIG. 11A shows original composite images ofFITC-dextran perfused cerebral microvessels. FIGS. 11B and C arecomputer generated three-dimensional images derived from the originalimages. Different colors in FIG. 11B represent individual vessels, whichwere not connected to each other. Green and red colors in FIG. 11C codefor diameter of blood vessels less then 7.5 μm (red) and larger then 7.5μm (green), respectively. Three-dimensional quantitative data revealedthat hMSCs with or without NONOate treatment significantly (p<0.05)increased numbers of branch points in the penumbra compared with numbersfound in the ipsilateral hemisphere of rats subjected to control MCAo.Segments of capillaries were significantly (p<0.05) shorter in theipsilateral hemisphere of the hMSC or/and NONOate treated group and thePBS control group than in the homologous tissue in the contralateralhemisphere, indicating that these are newly formed vessels after strokein ipsilateral hemisphere. Vascular diameter in the ipsilateral penumbraafter hMSCs treatment significantly (p<0.05) increased compared with thehomologous area of the contraiateral hemisphere and control MCAoanimals. Enlarged vessels can develop into capillaries after ischemia.Vessel surface area significantly (p<0.05) increased in hMSC with orwithout NONOate treated animals compared with control MCAO animals inthe ipsilateral hemisphere. Taken together, these data demonstrate thathMSCs with or without NONOate treatment enhances angiogenesis in theischemic brain. These data complement the BrdU angiogenesis data andindicate that combination therapy promotes angiogenesis.

The enhance induction of angiogenesis is also evident from in vitrostudies of tubule formation in brain derived endothelial cells. FIG. 12demonstrates that hMSC-supernatant (FIG. 12 b) and NONOate (FIG. 12 c)strongly induces endothelial tube formation by brain-derived endothelialcells compared with control medium (DMEM, FIG. 12 a). The endothelialcells formed a network of capillary-like structures with numerousintercellular contacts. Total tube length was significantly increased(p<0.01) in supernatant from cultural hMSCs (6.9±0.72 mm/mm²) andNONOate treatment (4.6±0.6 mm/mm²) compared with the control medium(DMEM, 1.4

0.1 mm/mm ). Total tube length was significantly increased insupernatant from cultural hMSCs compared with the NONOate. These datashow that both hMSCs and NONOate promote capillary-tube formation.

VEGF:

To obtain insight into the mechanisms associated with the induction ofangiogenesis and neurogenesis, the fact that combination therapy inducesthe expression of neurotrophic and growth factors in brain was tested.Data are presented on the levels of vascular endothelial growth factor(VEGF) in brain after treatment with MSCs, DETA-NONOate, combination(MSC+NONO) therapy, and control, PBS treated animals subjected to MCAo.FIG. 13 shows that using the Sandwich ELISA method, VEGF secretion fromendogenous cells (rat VEGF) was significantly increased in hMSCs withNONOate treatment groups compared with MCAo control group. Rat VEGFsecretion was borderline increased in hMSCs alone treatment group.Single dose NONOate alone treatment did not show a significant increasein VEGF compared with MCAo control group. These data indicate thatsubtherapeutic combination MSC+NONO therapy significantly enhances VEGFsecretion compared to individual therapy.

EXAMPLE 4

Induction of Cell Proliferation in Normal Monischemic Animals:

The effects of an NO donor administered to normal young adult rats onthe induction of cell proliferation within three regions of brain, thedentate gyrus, the olfactory bulb (OB) and the subventricular zone (SVZ)were tested. An NO donor,(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)aminio]diazen-1-ium-1,2-diolate(DETA/NONO-ate), was selected, because this compound is a highlyefficient NO donor with a half-life of 57 hours under physiologicalconditions (Beckman, 1995; Estevez et al., 1998). Young male Wister rats(3-4 month old) received 4 consecutive I.V bolus doses of DETA/NONOate(0.1 mg/kg each, every 15 min, and total dose of 0.4 mg/kg) on the firstexperimental day and DETA/NONOate (0.4 mg/kg) was administered (i.p)daily for an additional 6 consecutive days. Rats that received salinewere used as a control group. Bromodeoxyuridine (BrdU) was used asmitotic labeling to measure cell proliferation. Animals received dailyi.p injections of BrdU (50 mg/kg, Sigma) on the first experimental dayand subsequently for 14 consecutive days. Rats were sacrificed at 14 and42 days after the treatment. BrdU immunoreactive nuclei were counted ona computer monitor to improve visualization and in one focal plane toavoid over-sampling. Structures were sampled either by selectingpredetermined areas on each section (OB) or by analyzing entirestructures on each section (SVZ and dentate gyrus) (Zhang et al., 2001).All BrdU immunoreactive-positive nuclei in these areas are presented asthe number of the BrdU immunoreactive cells/mm2. Density for theselected several sections was averaged to obtain a mean density valuefor each animal (Zhang et al., 2001).

FIG. 15 shows the cell proliferation in the brain of the young adultrats, administered DETA/NONO-ate. Statistically significant increased inthe numbers of BrdU reactive cells were demonstrated within the dentategyrus (FIG. 15A), SVZ (FIG. 15B) and OB (FIG. 15C). More than 95% of thenewly generated cells within the dentate gyrus exhibited neuronalmarkers of NeuN and MAP2, indicating that these cells have the potentialto integrate into the tissue. The cells within the SVZ and the OB werenot characterized with double-labeled immunohistochemistry. However,morphologically, they resembled proliferating cells. The progenitorcells in the SVZ of the lateral ventricle migrate into the OB(Alvarez-Buylla et al., 2000). Thus, these data clearly indicate thatNO, which in the developing brain has been associated with cellproliferation and migration, induces cell proliferation and migration inthe adult brain. In these studies only cell proliferation was measuredin both groups of animals, and not the behavioral and functional effectsof cell proliferation. However, there are substantial supporting datathat cell proliferation within the dentate gyrus translates intoimproved learning in mice (Gould and Gross, 2002).

The question of whether this induction of neurogenesis by means of an NOdonor in young adult (3-4 month old) rats would also be present in olderrats was also tested. To test this hypothesis, 18-month-old male Wistarrats were treated with DETA/NONOate, using the identical experimentalprotocol described for young rats. FIG. 16 shows cell proliferation thethree regions, dentate gyrus (FIG. 16A), SVZ (FIG. 16B), OB (FIG. 16C),and described above for the young rats. As in the young rats, treatmentwith DETA/NONOate significantly increased the number of proliferatingcells. For the saline treated animals, the baseline cell proliferationwas reduced by approximately a factor of 2 in the SVZ and dentate gyrus.In the SVZ and dentate gyrus, treatment with DETA/NONOate increased cellproliferation in a similar ratio in the old as well as the younganimals. The relative increase in the number of proliferated cellswithin the OB was not as robust in the old animals as in the younganimals. This may be attributed to a loss of cell migration potential inthe old compared to the young animals.

These data provide novel and important observations. One is that cellproliferation can be induced in the old animals as in the young animals.The percent increase in proliferation is similar for the old and theyoung animals. However, very obvious, is the decease in the absolutenumbers of proliferating cell in the old versus the young rats.Functional correlates of cell proliferation in the old animals were notmeasured, and consequently we have no data on whether the increase incells within the dentate translates into improved function.

NO Donor Enhances Functional Recovery After Stroke:

It has been demonstrated that treatment of DETA/NONOate induces cellproliferation and neurogenesis in non-ischemic young rats as well as inyoung rats subjected to embolic stroke (Zhang et al., 2001). Thetreatment of rats initiated at one day after stroke translated intosignificant functional benefit. Thus, the data demonstrate that apharmacological agent that releases NO when administered to animals 1day after induction of a major ischemic stroke encompassing theterritory of the middle cerebral artery (MCA) improves functionaloutcome (Zhang et al., 2001).

The question arises as to the specificity of this NO agent for theinduction of neurogenesis and functional benefit. Are the effectsspecific for DETA/NONOate or would other agents that donate NO likewiseprovide functional benefit? To test this question, rats were treatedwith another NO donor, S-nitroso-N-acetylpenicillamine (SNAP, Sigma)that is structurally different from DETA/NONOate. Young male adultWistar rats were subjected to embolic MCA occlusion (Zhang et al.,1997). SNAP at a dose of 30 μg/kg was intravenously administered to ratsas a bolus followed by 300 μg/kg/h infusion for 60 minutes at 24 hoursafter embolic MCA occlusion. As functional outcome measures, the rotarodtest which assesses motor function such as coordination and balance(Zhang et al., 2000), the adhesive removal test which measures forelimbsomato-sensorimotor asymmetries (Schallert et al., 2000), and animalbody weight were measured prior to treatment and at 2, 4, 7, and 14 daysafter treatment. Animals were sacrificed at 14 days after stroke and theinfarct volume was measured (Zhang et al., 1997). FIG. 17 shows infarctvolume and the functional outcome measurements for the saline treatedand the SNAP treated groups. There was no significant difference involume of cerebral infarction between the treated and the non-controltreated groups (FIG. 17A).. However, significant improvement in functionmeasured with the rotarod (FIG. 17B) and the adhesive removal test (FIG.17C) was noted by 4 days after the onset of stroke. These benefitspersisted to the time of sacrifice at 14 days post stroke. Animal bodyweight (FIG. 17D), as an index of general physiological well being, wassignificantly increased compared to vehicle-saline treated animals at 7days after stroke. These data clearly demonstrate that treatment with anNO donor such as SNAP provides significant functional benefit to theanimals, without affecting the volume of cerebral infarction (FIG. 17A).Thus, the effect of the treatment is one of restorative therapy notneuroprotective therapy. These data indicate that pharmacologicalagents, such as NO donors, can enhance function after stroke. Functionalimprovement in these animals is associated with change and remodeling ofbrain. Neurogenesis and cell proliferation as well as angiogenesis andincreased levels of synaptic proteins are induced by the NO donormolecules.

NO is an activator of soluble guanylate cyclase and causes increasedcGMP in target cells (Ignarro, 1989; Garthwaite and Boulton, 1995). cGMPhas been associated with changes in axon extension and modification ofneuronal connections (Williams et al., 1994). It is possible that cGMPitself plays an important role in promoting brain plasticity. Increasebrain levels of cGMP in rats treated with NO donors indicate that the NOdonors enter the brain (Zhang et al., 2001). Another way to induce anincrease in cGMP in brain is to inhibit the activity of the enzyme thatbreaks down cGMP. Phosphodiesterase type 5 (PDE 5) enzyme is highlyspecific for hydrolysis of cGMP (Corbin and Francis, 1999; Kotera etal., 2000). One way therefore to reduce the breakdown of cGMP and henceto increase levels of cGMP in brain is to reduce or inhibit PDE 5. Totest the effect of administering a compound that inhibits PDE 5, adultmale rats were fed sildenafil (2 mg/kg) daily for 7 days at 24 hoursafter the onset of stroke. FIG. 18 shows the presence of PDE 5 in brain.Feeding the animals sildenafil significantly improved functionaloutcome, as measured in an array of functional outcome measurements(Zhang et al., 2002). This therapeutic benefit was evident without areduction of cerebral infarction, a similar condition observed withother NO donors. Thus, these data show that cGMP can be an importantmediator of brain plasticity after stroke. This plasticity can alsoimprove functional response.

In general, these data indicate that agents that affect NO and cGMP canalter normal, aged and injured brain. Not only is cell proliferation andangiogenesis increased, but also, significant functional benefit isobtained. There are other, cellular based ways to induce brainremodeling and functional improvement after stroke and neural injury.One approach, which has clinical implications, is to employ a populationof cells, such as bone marrow stromal cells. These cells whenadministered to rodents enter brain and evoke the production of avariety of neurotrophic factors and cytokines that remodel brain andprovide significant functional benefit (Review, Chopp and Li, 2002).

FIG. 15 includes bar graphs that show the number of BrdU immunoreactivecells in the dentate gyrus (FIG. 15A), in the SVZ (FIG. 15B), and in theOB (FIG. 15C) in non-ischemic young adult rats at 14 (

) and 42 (

) days after treatment with DETA/NONOate or saline. *p<0.05 and **p<0.01versus the saline treated group.

FIG. 16 includes bar graphs that show the number of BrdU immunoreactivecells in the dentate gyrus (FIG. 16A), in the SVZ (FIG. 16B), and in theOB (FIG. 16C) in non-ischemic aged rats at 14 (

) and 42 (

) days after treatment with DETA/NONOate or saline. *p<0.05 and **p<0.01versus the saline treated group.

FIG. 17 shows the effect of SNAP treatment on infarct volume (FIG. 17A),rotarod (FIG. 17B) and adhesive removal (FIG. 17C) tests as well asanimal body weight (FIG. 17D). *p<0.05 and **p<0.01 versus the salinetreated group. n=8 for each group.

FIG. 18 shows RT-PCR of PDE5A1 (FIG. 18A) and PDE5A2 (FIG. 18B) mRNA inthe cortex of non-ischemic rats (N in FIG. 18A and FIG. 18B) and theipsilateral cortex of rats 2 hours to 7 days after ischemia. M=marker,N=non-ischemic rats, 2 hours, 4 hours, 1 day, 2days and 7days =timesafter ischemia.

Conclusion:

It has been demonstrated that a pharmacological therapy based on NO andcGMP induces changes in brain that enhance restoration of function afterstroke, and induce cell proliferation and neurogenesis in the normalyoung and old animal. These data along, with other studies on promotionof brain plasticity using cell-based therapy, opens new opportunities totreat neurodegenerative disease and neural injury.

Throughout this application, various publications, including UnitedStates patents, are referenced by author and year and patents by number.Full citations for the publications are listed below. The disclosures ofthese publications and patents in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used is intended to bein the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the described invention, theinvention may be practiced otherwise than as specifically described.

References

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1. A method of promoting neurogenesis comprising the step of:administering a therapeutic amount of a phosphodiesterase inhibitorcompound to a patient in need of neurogenesis promotion.
 2. The methodaccording to claim 1, further including administering cellular therapyto the patient.
 3. A compound for promoting neurogenesis comprising aneffective amount of a phosphodiesterase inhibitor sufficient to promoteneurogenesis.
 4. The compound according to claim 3, further including acellular therapy.
 5. A neurogenesis promoter comprising aphosphodiesterase inhibitor in a pharmaceutically acceptable carrier. 6.The neurogenesis promoter according to claim 5, wherein saidphosphodiesterase inhibitor augments nitric oxide in a tissue.
 7. Theneurogenesis promoter according to claim 6, wherein saidphosphodiesterase inhibitor is sildenafil.
 8. A method of augmenting theproduction of neurons by administering an effective amount of aphosphodiesterase inhibitor to a site in need of augmentation.
 9. Themethod according to claim 8, further including administering cellulartherapy to the site.
 10. A method of increasing neurological function byadministering an effective amount of a phosphodiesterase inhibitor to apatient.
 11. The method according to claim 10, further includingadministering cellular therapy to the patient.
 12. A method ofincreasing cognitive and neurological function by administering aneffective amount of a phosphodiesterase inhibitor compound to a patient.13. The method according to claim 12, further including administeringcellular therapy to the patient.